Transporter genes

ABSTRACT

Novel genes significantly homologous to organic cation transporters OCT1 and OCT2 have been successfully isolated by screening a fetal gene library by random sequencing. Proteins encoded by these genes function as transporters of various organic cations.

[0001] This application is a continuation-in-part of PCT/JP98/04009filed Sep. 7, 1998, and claims priority from Japanese Application No.9/260972, filed Sep. 8, 1997 and Japanese application Ser. No.10/156,660, filed May 20, 1998.

FIELD OF THE INVENTION

[0002] The present invention relates to transporters, proteins involvedin transport of substances from the outside to the inside of cells orvice versa.

BACKGROUND OF THE INVENTION

[0003] Recently, the involvement of various transporters localized onthe plasma membrane in the uptake system for nutrients and endogenoussubstances into cells and their transport mechanisms have been clarified(Tsuji, A. and Tamai, I., Pharm. Res., 13, 963-977, 1996). Thesetransporters recognize the structures of substances to be transported toselectively transport specific substances. Transporters that recognizethe relatively wide range of structures may possibly recognize foreignsubstances such as drugs by mistake, and actively take in them intocells. It is believed that drugs permeate through the plasma membranefundamentally by simple diffusion depending on their physicochemicalproperties such as molecular size, hydrophobicity, and hydrogen-bindingcapacity. Particularly, in the case of ionic drugs, only molecules inthe non-dissociated form can permeate through the plasma membraneaccording to the pH partition hypothesis. However, it has become evidentthat a number of drugs penetrate through the cell membrane by a specificmechanism other than simple diffusion, that is, an active transportmediated by transporters, in organs that require efficient exchange ofintracellular and extracellular substances, including small intestine,uriniferous tubule, placenta, epithelial cells of choroid plexus,hepatocytes, and blood-brain barrier (Tamai, I. and Tsuji, A.,Pharmacia, 31, 493-497, 1995; Saito, H. and Inui, K., Igaku no Ayumi,179, 393-397, 1996; Tamai, I., Yakubutsu Dotai (Pharmacokinetics), 11,642-650, 1996). For example, it is known that although oral β-lactamantibiotics of the non-esterified type are amphoteric or negativelycharged in physiological pHs and sparingly soluble in fat, they arereadily absorbed through the intestine. The transport study using theisolated membrane-vesicles system demonstrated that an H³⁰-drivenpeptide transporter localized on the brush-border-is involved in theabsorption process of these drugs (Tsuji, A. et al., J. Pharmacol. Exp.Ther., 241, 594-601, 1987). Although the specificity of a peptidetransport system in terms of the peptide size is so strict as torecognize di- or tri-peptides but not tetrapeptides or larger peptides,it has a rather broad substrate specificity to recognize peptidescomprising non-natural amino acids. The peptide transporter mediatestransport of β-lactam antibiotics mistakenly due to its broad substratespecificity. This property has been unexpectedly utilized in theclinical field (Tsuji, A., American Chemical Society (eds. Taylor, M.D., Amidon, G. L.), Washington, D.C., 101-134, 1995). Furthermore, apossibility that a transporter is also involved in permeation ofsubstances with a high hydrophobicity such as fatty acids through theplasma membrane has been reported (Schaffer, J. and Lodish, H., Cell,79, 427-436, 1994).

[0004] Since various transporters are supposed to be distributed inorgans and cells based on the physiological roles of the organs andcells, their distribution and functions may be specific to organs.Therefore, transporters are expected to be used to impart an organspecificity to pharmacokinetics. In other words, an organ-specific drugdelivery system (DDS) can be constructed utilizing transporters. If drugabsorption solely relied on simple diffusion is improved by elevatingits hydrophobicity, an effect of the drug obtained in the initialtransport in the liver can be enhanced and the drug can non-specificallytranslocates into any organ. In addition, it would also be possible toincrease the drug absorption independently of its fat-solubility bydesigning the drug utilizing the substrate specificity of transporters(Hayashi, K. et al., Drug Delivery System, 11, 205-213, 1996). For thispurpose, it is necessary to identify various transporters at themolecular level and analyze their properties in detail. However, theirmolecular level identification are greatly behind studies on theirmembrane physiology because they are difficult to handle biochemicallyand require complicated processes in their functional assays.

[0005] Recently, cDNAs of several transporters have been cloned by theexpression cloning method using Xenopus oocytes, a foreign geneexpression system, and structural homology among them has been revealed(Fei, Y.-J. et al., Nature, 368, 563-566, 1994). For example, Koepsellet al. cloned an organic cation transporter, OCT1, which is assumed tobe localized on a basement membrane, using the expression cloning methodin 1994 (Grundemann, D. et al., Nature, 372, 549-552, 1994).Subsequently, OCT2 was identified by homology cloning based on thesequence of OCT1 (Okuda, M. et al., Biochem. Biophys. Res. Commun., 224,500-507, 1996). OCT1 and OCT2 show homology as high as 67% to each other(Grundemann, D. et al., J. Biol. Chem., 272, 10408-10413, 1997). Both ofthem are intensely expressed in the kidney, but differ in the organdistribution; OCT1 is also expressed in the liver, colon, and smallintestine, while OCT2 expression is specific to the kidney.

[0006] Only a few reports on identification of transporters at themolecular level, including the reports, are available, and there wouldbe many unknown transporters that may be clinically useful.

SUMMARY OF THE INVENTION

[0007] An object of this invention is to provide a family of noveltransporter genes, proteins encoded by these genes, and their use.

[0008] The present inventors have screened a fetal gene libraryconstructed using the subtractive method by random sequencing based on aworking hypothesis that fetal genes include those which are involved invarious disorders including cancer and are specifically or intenselyexpressed in fetal tissues. The inventors discovered an unknown geneshowing a significant homology with those for organic cationtransporters, OCT1 and OCT2, and attempted to isolate this gene, whichwas assumed to encode a novel transporter. Thus, the inventors succeededin isolating the desired gene by screening a cDNA library derived fromhuman fetus. Furthermore, the inventors studied the transporter activityof a protein encoded by the isolated human gene and found that theprotein, in fact, functioned as a transporter for various organiccations. The inventors also succeeded in isolating a mouse genecorresponding to the isolated human gene.

[0009] This invention relates to a family of novel transporter genes,proteins encoded by these genes, and their use, and more specificallyto:

[0010] (1) a protein comprising an amino acid sequence set forth in SEQID NOs: 1, 3, 22, or 27, or a protein comprising said amino acidsequence in which one or more amino acid residues are substituted,deleted, or added, and having an activity to transport an organiccation;

[0011] (2) a protein encoded by a DNA hybridizing to a DNA comprisingnucleotide sequence according to SEQ ID NOs: 2, 4, 23, or 28, and havingan activity to transport an organic cation;

[0012] (3) a DNA encoding the protein according to (1) or (2);

[0013] (4) a vector comprising the DNA according to (3);

[0014] (5) a transformant expressibly carrying the DNA according to (3);

[0015] (6) a method for producing the protein according to (1) or (2),the method comprising culturing the transformant according to (5);

[0016] (7) an antibody that binds to the protein according to (1) or(2); and

[0017] (8) a DNA specifically hybridizing to a DNA comprising anucleotide sequence set forth in SEQ ID NOs: 2, 4, 23, or 28, andconsisting of at least 15 nucleotides.

[0018] Nucleotide sequences of cDNAs of novel human transportersisolated by the present inventors are shown in SEQ ID NO: 2 (designatedas “human OCTN1”) and SEQ ID NO: 4 (designated as “human OCTN2”),respectively. Amino acid sequences of proteins encoded by these cDNAsare shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. Amino acidsequences of these two proteins included in the transporter proteins ofthis invention showed such a high overall homology as about 76%, andboth of them retained the following consensus sequence which isconserved in various types of transporters including the glucosetransporter: [Leu, Ile, Val, Met, Ser, Thr, Ala, Gly]-[Leu, Ile, Val,Met, Phe, Ser, Ala, Gly]-Xaa<2>-[Leu, Ile, Val, Met, Ser, Ala]-[Asp,Glu]-Xaa-[Leu, Ile, Val, Met, Phe, Tyr, Trp, Ala]-Gly-Arg-[Arg,Lys]-Xaa<4-6>-[Gly, Ser, Thr, Ala] (Maiden, M. C. et al., Nature, 325,641-643, 1987). In fact, these proteins have an activity to transportvarious organic cations (see Examples 6 to 8).

[0019] The present inventors also isolated mouse genes corresponding tothe above-described human OCTN1 and human OCTN2. Nucleotide sequences ofthe isolated cDNAs are shown in SEQ ID NO: 23 (designated as “mouseOCTN1”) and SEQ ID NO: 28 (designated as “mouse OCTN2”), respectively.Amino acid sequences of proteins encoded by these cDNAs are shown in SEQID NOs: 22 and 27, respectively.

[0020] Transporter proteins of this invention also include those havingthe additional activity to transport substances other than organiccations as far as they retain the organic cation transport activity.Organic cations include, for example, TEA, carnitine, quinidine, andpyrilamine, but are not limited to them. They also include carcinostaticagents such as actinomycin D, etoposide, vinblastine, daunomycin, etc.Transporter proteins of this invention include those having the activityto transport organic cations not only from the outside to the inside ofcells but also from the inside to the outside of cells.

[0021] Transporter proteins of this invention can be prepared asrecombinant proteins using recombination techniques or natural proteins.Recombinant proteins can be prepared, for example, as described below,by culturing cells transformed with DNA encoding proteins of thisinvention. Natural proteins can be isolated from the kidney and cancercell strains such as Hela S3, which highly express the proteins of thisinvention, by the method well known to those skilled in the art, forexample, affinity chromatography using an antibody of this inventiondescribed below. The antibody may be either polyclonal or monoclonal. Apolyclonal antibody can be prepared by purifying serum obtained from,for example, a small animal such as a rabbit immunized with proteins ofthis invention by known methods, for example, ammonium sulfateprecipitation, protein A or protein G column, DEAE-ion exchange columnchromatography, affinity column chromatography coupled with the proteinof this invention, etc. A monoclonal antibody can be prepared byimmunizing a small animal such as a mouse with the protein of thisinvention, excising the spleen from the mouse, grinding the tissue intocells, fusing them with mouse myeloma cells using a fusing agent such aspolyethylene glycol, and selecting a clone that produces an antibody toproteins of this invention out of fused cells (hybridomas) thusproduced. Then, hybridomas thus selected are transplanted into theabdominal cavity of a mouse, and the ascites is collected from themouse. A monoclonal antibody thus obtained can be purified by, forexample, ammonium sulfate precipitation, protein A or protein G column,DEAE-ion exchange column chromatography, affinity column chromatographycoupled with the protein of this invention, etc. When the antibody thusobtained is administered to human subjects, a humanized antibody or ahuman antibody is advantageously used to reduce the immunogenicity. Anantibody can be humanized by, for example, the CDR grafting methodcomprising cloning an antibody gene from monoclonal antibody-producingcells and grafting the epitope portion thereof into an existing humanantibody. A human antibody can be prepared by the usual method forpreparing a monoclonal antibody except for immunizing a mouse whoseimmune system is replaced with the human's.

[0022] The term “substantially pure” as used herein in reference to agiven polypeptide means that the polypeptide is substantially free fromother biological macromolecules. The substantially pure polypeptide isat least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight.Purity can be measured by any appropriate standard method, for example,by column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

[0023] A “conservative amino acid substitution” is one in which an aminoacid residue is replaced with another residue having a chemicallysimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

[0024] It is also possible for those skilled in the art to prepareproteins having functions equivalent to the transporter proteins of thisinvention (human OCTN1, human OCTN2, mouse OCTN1, and mouse OCTN2) byappropriately modifying amino acid residues of the proteins by, forexample, substitution, using well known methods. Mutation of amino acidsof the proteins can occur also spontaneously. Such mutant proteins whichare obtained by altering the amino acid sequence of the transporterproteins of this invention by substitution, deletion, or addition ofamino acid residues, and are functionally equivalent to those of thetransporter proteins are also included in the proteins of thisinvention. Herein, “functionally equivalent” means that proteins have anactivity to transport organic cations. Methods well known to thoseskilled in the art for altering amino acids include, for example, thesite-specific mutagenesis system by PCR (GIBCO-BRL, Gaithersburg, Md.),site-specific mutagenesis by oligonucleotide (Kramer, W. and Fritz, H.J. (1987) Methods in Enzymol., 154: 350-367), Kunkel's method (MethodsEnzymol., 85, 2763-2766 (1988)), etc. The number of amino acids that canbe substituted is usually 10 amino acid residues or less, preferably 6or less, and more preferably 3 or less. The site of substitution,deletion, or addition of amino acid residues is not particularly limitedas far as the activity of proteins of this invention is retained. It ispossible to detect the transporter activity of proteins, for example, bythe method described below in Example 6.

[0025] It is routine for those skilled in the art to obtain proteinsfunctionally equivalent to the transporter proteins of this invention byisolating and using DNAs highly homologous to the DNA sequences encodingthe transporter proteins of this invention (human OCTN1, human OCTN2,mouse OCTN1, and mouse OCTN2) or portions thereof using hybridizationtechniques (Sambrook, J. et al., Molecular Cloning 2nd ed., 9.47-9.58,Cold Spring Harbor Lab. press, 1989), etc. These proteins functionallyequivalent to those of transporter proteins of this invention are alsoincluded in proteins of this invention. Here, “functionally equivalent”means that proteins have an activity to transport organic cations. DNAsthat hybridize to the DNAs encoding the proteins of this invention canbe isolated from other organisms, for example, rats, rabbits, cattle,etc. as well as humans and mice. Especially, tissues such as the kidneyare suitable as sources of such DNAs. These DNAs isolated usinghybridization techniques usually have a high homology with theabove-described DNAs encoding the transporter proteins of thisinvention. “High homology” means at least 70% or more, preferably atleast 80% or more, and more preferably at least 90% or more of aminoacid sequence identity. The “percent identity” of two amino acidsequences or of two nucleic acids is determined using the algorithm ofKarlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990),modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA90:5873-5877, 1993). Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al. (J. Mol. Biol: 215:403-410,1990). BLAST nucleotide searches are performed with the NBLAST program,score=100, wordlength=12. BLAST protein searches are performed with theXBLAST program, score=50, wordlength=3. Where gaps exist between twosequences, Gapped BLAST is utilized as described in Altschul et al.(Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

[0026] One example of hybridization conditions for isolating such DNAsis as follows. That is, after the pre-hybridization at 55° C. for 30 minor more in the “ExpressHyb Hybridization Solution” (CLONTECH), a labeledprobe is added, and hybridization is performed by heating the reactionmixture at 3° C. to 55° C. for 1 h or more. Then, the reaction productis successively washed in 2×SSC and 0.1% SDS three times at roomtemperature for 20 min, and then in 1×SSC and 0.1% SDS once at 37° C.for 20 min. More preferable conditions are as follows. After thepre-hybridization at 60° C. for 30 min or more in the “ExpressHybHybridization Solution” (CLONTECH), a labeled probe is added, andhybridization is performed by heating the reaction mixture at 60° C. for1 h or more. Then, the reaction product is successively washed in 2×SSCand 0.1% SDS three times at room temperature for 20 min, and then in1×SSC and 0.1% SDS twice at 50° C. for 20 min. Still more preferableconditions are as follows. After pre-hybridization at 68° C. for 30 minor more in the “ExpressHyb Hybridization Solution” (CLONTECH), a labeledprobe is added, and hybridization is performed by heating the reactionmixture at 68° C. for 1 h or more. Then, the reaction product issuccessively washed in 2×SSC and 0.1% SDS three times at roomtemperature for 20 min, and then in 0.1×SSC and 0.1% SDS twice at 50° C.for 20 min.

[0027] The present invention also relates to DNAs encoding theabove-described transporter proteins of this invention. DNAs of thisinvention may be cDNA, genomic DNAs, and synthetic DNAs. The DNAs of thepresent invention can be used for producing proteins of this inventionas recombinant proteins. That is, it is possible to prepare proteins ofthis invention as recombinant proteins by inserting DNAs encodingproteins of this invention (e.g. DNAs comprising the nucleotidesequences set forth in SEQ ID NOs: 2, 4, 23, and 28) into an appropriateexpression vector, culturing transformants obtained by transfectingsuitable cells with the vector, and purifying the proteins thusexpressed. Cells to be used for producing recombinant proteins include,for example, mammalian cells such as COS cells, CHO cells, NIH3T3 cells,etc., insect cells such as Sf9 cells, yeast cells, E. coli, and so on.Vectors used for the intracellular expression of recombinant proteinsvary depending on host cells, including, for example, pcDNA3(Invitrogen), pEF-BOS (Nucleic Acids Res., 1990, 18(7), p5322), etc. formammalian cells, “BAC-to-BAC baculovirus expression system” (GIBCO BRL),etc. for insect cells, “Pichia Expression Kit” (Invitrogen), etc. foryeast cells, pGEX-5×-1 (Pharmacia), “QIAexpress system” (Qiagen), etc.for E. coli. Host cells can be transformed with vectors, for example, bythe calcium phosphate method, the DEAE-dextran method, the method usingcationic liposome DOTAP (Boehringer Mannheim), the electroporationmethod, the calcium chloride method, etc. Recombinant proteins can bepurified from recombinants thus obtained using standard methods, forexample, as described in “The Qiaexpressionist Handbook, Qiagen, Hilden,Germany.”

[0028] The present invention also relates to DNAs consisting of at least15 nucleotides that specifically hybridize to the DNAs encoding proteinsof this invention. Herein, “specifically hybridize” means that a DNAdoes not cross-hybridize to other DNAs encoding other proteins underusual hybridization conditions, preferably under the stringenthybridization conditions. Such a DNA can be utilized as a probe fordetecting and isolating DNA encoding the protein of this invention, andas a primer for amplifying the DNA. By hybridization under “stringentconditions” is meant hybridization at 37° C., 1×SSC, followed by washingat 42° C., 0.5×SSC.

[0029] An “isolated nucleic acid” is a nucleic acid the structure ofwhich is not identical to that of any naturally occurring nucleic acidor to that of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in mixtures of different (i) DNA molecules, (ii)transfected cells, or (iii) cell clones: e.g., as these occur in a DNAlibrary such as a cDNA or genomic DNA library.

[0030] The transporter proteins of this invention can be used to controlinternal absorption and dynamics of drugs. Based on the results ofdetailed analysis of the substrate specificity of transporter proteinsof this invention, drugs can be designed so as to be transported bythese transporters and absorbability of the drugs mediated by thesetransporter proteins can be improved. Conventional modifications toenhance hydrophobicity are no longer necessary for drugs so designed,which enables speedily and efficiently developing water-soluble drugsthat are easy to handle. The drugs thus developed is thought to beabsorbed principally depending on the internal distribution pattern oftransporter proteins of this invention, and an organ-specific deliveryof the drugs thus becomes possible. Especially, if the transporterproteins of this invention are distributed in the target organ of adrug, an ideal drug delivery system (DDS) can be developed. If a drug isto be absorbed mediated by not the transporter proteins of thisinvention but other transporters, the drug can be designed so as to bespecific to other transporter proteins by designing it considering thesubstrate specificity of the transporter proteins of this invention.Since the transporter proteins of this invention are present in thekidney, it is possible to reduce the nephrotoxicity produced by a drugby designing the drug so that it can be readily excreted by thetransporter proteins of this invention.

[0031] Another possible application of this invention is to develop adrug targeting the transporter proteins of this invention. Thetransporters play important roles in the absorption mechanism ofnutrients and drugs, or the excretion mechanism of drugs and internalmetabolites. Thus, damage or abnormal elevation of the transporter'sfunctions may cause some disorders. It is considered to be efficaciousagainst such disorders to administer a drug containing a compound thatinhibits or enhances functions of the transporter proteins of thisinvention, or regulates the expression level of the transporter gene ofthis invention and the amount of the transporter proteins. The DNAs ofthis invention can be used in gene therapy for disorders caused byabnormalities in the activity and expression of the proteins of thisinvention. In this case, the DNA of this invention are inserted to anadenovirus vector (e.g. pAdexLcw), a retrovirus vector (e.g. pZIPneo),etc., and administered into the body by either ex vivo method or in vivomethod. Gene therapy can also be performed by administering a syntheticantisense DNA to the body either directly or after inserted into theabove-described vector.

[0032] Especially, since “OCTN2” included in the transporter proteins ofthis invention efficiently transports carnitine, chemotherapy withcompounds to control the activity of “OCTN2” or gene therapy using the“OCTN2” gene is considered to be efficacious against variouspathological conditions such as fatty liver, myocardiopathy, myopathy,etc. caused by hypocarnitinemia.

[0033] The transporter proteins of this invention are expressed in avariety of cancer cell strains, which suggests that the proteins maytransport drugs into tumor cells. If this is the case, it is possible todevelop carcinostatics that will be readily absorbed mediated by thetransporter proteins of this invention. On the contrary, mechanisms totransport and excrete substances by the transporter proteins of thisinvention may function to excrete carcinostatics in tumor cells so thatthe cells acquire resistance to drugs. If the transporter proteins ofthis invention are involved in a mechanism of tumor cells to acquiredrug resistance, a carcinostatic effect can be enhanced by a combineduse of inhibitors of the transporter proteins of this invention withcarcinostatics.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 represents hydrophobicity plots of human OCTN1 and humanOCTN2 according to Kyte & Doolittle's calculating formula with a windowof nine amino acid residues. Numerals on the plots indicate putativetransmembrane regions.

[0035]FIG. 2 represents electrophoretic patterns showing the results ofNorthern blot analysis of human OCTN1.

[0036]FIG. 3 compares the amino acid sequence of human OCTN1 with thatof human OCTN2. Amino acid residues conserved in both transporters areshaded. Sequences coinciding with the consensus sequences of sugartransporter and the ATP/GTP binding site are indicated by “+” and “*,”respectively.

[0037]FIG. 4 represents electrophoretic patterns showing the results ofNorthern blot analysis of human OCTN2.

[0038]FIG. 5 is a graph showing the TEA-absorbing activity of humanOCTN1. Clear circles represent untreated cells, and solid circlesrepresent human OCTN1-transfected cells.

[0039]FIG. 6 is a graph showing effects of the cold TEA added in theexperimental system in FIG. 5. In this graph, solid circles representhuman OCTN-transfected cells, and clear circles represent cellscontaining the vector with no insert. Clear triangles indicate the netuptake induced by human OCTN1 obtained by subtracting the clear circlevalues from the corresponding solid circle values.

[0040]FIG. 7 is a graph showing TEA concentration-dependency of theTEA-absorbing activity of human OCTN1.

[0041]FIG. 8 is a bar graph showing the activity of the humanOCTN1-transfected cells to absorb substances other than TEA.

[0042]FIG. 9 is a bar graph showing the results of transport experimentsusing Xenopus oocytes. Bars indicated with “OCTN1” and “Water” representthe uptake activity of the human OCTN1-injected cRNA oocytes and that ofthe water-injected oocytes (containing no cRNA), respectively. Uptakesof TEA, carnitine, mepyramine, quinidine, and actinomycin D wereobserved in human OCTN1 cRNA-injected oocytes, whereas water-injectedoocytes (containing no cRNA) exhibited almost no uptake activity.

[0043]FIG. 10 is a bar graph showing the results of transportexperiments for carcinostatics in Xenopus oocytes. Bars indicated with“OCTN1” and “Water” represent the uptake activity of the human OCTN1cRNA-injected oocytes and that of the water-injected oocytes (containingno cRNA), respectively. Uptakes of actinomycin D, etoposide,vinblastine, and daunomycin were observed in the human OCTN1cRNA-injected oocytes.

[0044]FIG. 11 is a bar graph showing the results of transportexperiments with human OCTN1 and human OCTN2 in HEK293 cells. HumanOCTN1 has the efficient transport activity for TEA and human OCTN2 forcarnitine.

[0045]FIG. 12 is a graph showing the results of Na⁺-dependency of thecarnitine transport activity of human OCTN2. Human OCTN2 exhibits atime-dependent carnitine transport activity (clear circle) in thepresence of Na⁺, while no such activity in the absence of Na⁺ (solidcircle), indicating that the carnitine transport activity of human OCTN2depends on the presence of Na⁺.

[0046]FIG. 13 shows the expression of mouse OCTN1 and mouse OCTN2 genesdetected by RT-PCR amplification in each tissue. G3PDH serves as acontrol, indicating that the amount of cDNA in each tissue is uniform.

DETAILED DESCRIPTION

[0047] The present invention is described below in more detail withreference to examples, but is not construed being limited thereto.

EXAMPLE 1 Construction of a Subtraction Library

[0048] A subtraction library was constructed using the PCR-Select™ cDNASubtraction Kit (CLONTECH) principally according to the method of LudaDiatchenko (Diatchenko, L. et al., Proc. Natl. Acad. Sci. USA, 93,6025-6030, 1996).

[0049] First, double-stranded cDNAs were synthesized from poly(A)+ RNAsderived from human fetal liver and adult liver by the standard methodusing MMLV reverse transcriptase. These cDNAs were blunt-ended with T4DNA polymerase and cleaved with RsaI. A part of the cDNAs derived fromfetal liver (tester) was divided in two portions, and they wereseparately ligated to two different adapters, adapter 1 and adapter 2,respectively (Table 1). A 120-fold excess of cDNA derived from adultliver (driver) was added to each of the above-described tester samples.The mixture was heat-denatured and subjected to the primaryhybridization at 68° C. for 8 h. After these two reaction mixtures fromthe primary hybridization were mixed together without heat-denaturation,an excessive amount of the heat-denatured driver was further addedthereto, and the mixture was subjected to the secondary hybridization at68° C. for about 16 h. The resulting reaction solution was diluted witha dilution buffer and incubated at 75° C. for 7 min. After the shorterstrands of adapters were removed, the reaction solution was used as atemplate for PCR. PCR using primers 1 (5′-CTAATACGACTCACTATAGGGC-3′₁,SEQ ID NO: 5) and 2 (5′-TGTAGCGTGAAGACGACAGAA-3′, SEQ ID NO: 6)corresponding to the adapters selectively amplified only cDNAs havingdifferent adapters at their both ends (subtracted cDNAs)(suppressionPCR). PCR was carried out using a portion of the resulting cDNA as atemplate, and nested PCR primers 1 (5′-TCGAGCGGCCGCCCGGGCAGGT-3′, SEQ IDNO: 7) and 2 (5′-AGGGCGTGGTGCGGAGGGCGGT-3′, SEQ ID NO: 8), which arefurther inwardly located from the PCR primers 1 and 2, to obtainproducts with further elevated selectivity. PCR products thus obtainedwere purified using the QIAquick PCR Purification kit (QIAGEN), andcloned into the pT7Blue-T vector (Novagen) by the TA cloning method toconstruct a subtraction library. TABLE 1 Adapter5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′ 1 3′-GGCCCGTCCA-5′Adapter 5′-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3′ 23′-GCCTCCCGCCA-5′

[0050] The longer strand of the partially single stranded DNA ofAdapters 1 and 2 are designated SEQ ID NOs:29 and 30, respectively, andthe shorter strand of Adapters 1 and 2 are designated SEQ ID NOs:31 and32, respectively.

EXAMPLE 2 CDNA Cloning

[0051] To analyze fetal genes, the subtraction library derived from thefetal liver was screened by random sequencing. Homology search (Blastx)of Expressed Sequence Tags (ESTs) thus obtained found a clone, OCTN1(fls 631) (292 bp) encoding amino acid sequence having significanthomology with the known organic cation transporters, OCT1 (Grundemann,D. et al., Nature, 372, 549-552, 1994) and OCT2 (Okuda, M. et al.,Biochem. Biophys. Res. Commun., 224, 500-507, 1996). Since the sequenceof this clone was novel and assumed to be a fragment derived from a newtransporter gene, cDNA comprising the whole open reading frame (ORF) ofthis gene was cloned.

[0052] The human fetal liver 5′-stretch cDNA library (CLONTECH) wasscreened using the original OCTN1 clone obtained from the subtractionlibrary derived from fetal liver as a probe. An insert of the originalOCTN1 clone was amplified by PCR using M13 P4-22 and M13 P5-22, andlabeled with [α-³²P]dCTP by the random primer method using the Ready-toGo DNA labeling beads (Pharmacia) to serve as a probe. Hybridization wascarried out at 68° C. in the ExpressHyb Hybridization Solution(CLONTECH) according to the method recommended by the manufacturer.Final washing was performed at 5° C. in 0.1×SSC and 0.1% SDS. Screeningabout 5×10 phage clones finally isolated seven positive clones cDNAinserts of these clones were amplified by PCR using vector primersdesigned based on a sequence of the λgt10 vector (GT10 S15′-CTTTTGAGCAAGTTCAGCCT-3′, SEQ ID NO: 9, and GT10 Al5′-AGAGGTGGCTTATGAGTATTTCTT-3′, SEQ ID NO: 10), or primers designedbased on the decoded cDNA sequences. The PCR products thus obtained weredirectly sequenced to determine the nucleotide sequences. Some regionsthat were difficult to be amplified were subjected to PCR using 7-deazadGTP as a substrate base (McConlogue, L. et al., Nucleic Acids Res., 16,9869, 1988).

[0053] Sequencing of cDNA inserts of these clones revealed that thehuman OCTN1 gene contains an ORF encoding a protein consisting of 551amino acid residues (putative molecular weight of about 62,000). Database search using this whole amino acid sequence confirmed that it has asignificant overall homology (about 34%) with OCT1 and OCT2.Hydrophobicity profile of this sequence obtained by Kyte & Doolittle'scalculating formula (Kyte, J. and Doolittle, R. F., J. Mol. Biol., 157,105-132, 1982) very closely resembled those of OCT1 and OCT2, indicatingthat the sequence has eleven to twelve putative transmembranehydrophobic regions (FIG. 1). This sequence contained one consensussequence of sugar transporter, ([Leu, Ile, Val, Met, Ser, Thr, Ala,Gly]-[Leu, Ile, Val, Met, Phe, Ser, Ala, Gly]-Xaa<2>-[Leu, Ile, Val,Met, Ser, Ala]-[Asp, Glu]-Xaa-[Leu, Ile, Val, Met, Phe, Tyr, Trp,Ala]-Gly-Arg-[Arg, Lys]-Xaa<4-6>-[Gly, Ser, Thr, Ala]), (160 to 175).This consensus sequence is present in the glucose transporters GLUT1 toGLUT7 in mammalian cells, and also present in various types oftransporters other than glucose transporters (Maiden, M. C. et al.,Nature, 325, 641-643, 1987). Furthermore, putative N-linkedglycosylation sequences (N-X-[ST]) were found in the amino acid sequenceof human OCTN1 at four sites (57 to 59, 64 to 66, 91 to 93, and 304 to306), and also five putative protein kinase C phosphorylation sites([ST]-X-[RK]) (164 to 166, 225 to 227, 280 to 282, 286 to 288, and 530to 532). In addition, the consensus sequence ([Ala,Gly]-Xaa(4)-Gly-Lys-[Ser, Thr]) of the ATP/GTP binding site is alsofound. This consensus sequence of the ATP/GTP binding site is alsopresent in the ATP binding protein or GTP binding protein, such askinases and ras family proteins, and that ATP or GTP binds to this site(Walker, J. E. et al., EMBO J., 1, 945-951, 1982). This sequence ispresent in the so-called ATP Binding Cassette (ABC) type transporter,and involved in the substance transport using the energy generated byhydrolysis of ATP (Higgins, C. F. et al., J. Bioenerg. Siomembr., 22,571-592, 1990; Urbatsch, I. L. et al., J. Biol. Chem., 270, 26956-26961,1995). Presence of this consensus sequence indicates that OCTN1 proteinmay be an ATP or GTP-dependent transporter.

[0054] Nucleotide sequencing was performed by the cycle-sequencingmethod with a plasmid DNA prepared by the alkaline-SDS method or a PCRproduct obtained by colony PCR, etc. as a template using the ABI PRISM™Dye Terminator Cycle Sequencing Ready Reaction Kit With AmplyTaq DNAPolymerase, FS, followed by decoding with the ABI 377 DNA Sequencer(Perkin Elmer). Colony PCR was carried out by directly suspending acolony of a recombinant in a PCR reaction solution containing vectorprimers M13 P4-22 (5′-CCAGGGTTTTCCCAGTCACGAC-3′, SEQ ID NO: 11) and M13P5-22 (5′-TCACACAGGAAACAGCTATGAC-3′, SEQ ID NO: 12). After thecompletion of PCR, a DNA insert thus amplified was separated fromunreacted primers and nucleotides by gel filtration, etc. to serve as atemplate for sequencing.

EXAMPLE 3 Northern Analysis

[0055] Distribution of human OCTN1 in tissues was investigated byNorthern analysis (FIG. 2). A 3′-end fragment of human OCTN1 (the latterhalf from around the base 1,100) was labelled with [α−³²P]dCTP by therandom primer method using the Ready-to Go DNA labeling beads(Pharmacia) to serve as a probe. Hybridization was performed using theMultiple Tissue Northern (MTN) Blot-Human, Human III, Human IV, HumanFetal II, and Human Cell lines (CLONTECH) at 68° C. in the ExpressHybHybridization Solution (CLONTECH) according to the method recommended bythe manufacturer. Final washing was performed at 50° C. in 0.1×SSC and0.1% SDS. As a result, RNA of about 2.5 kb was strongly expressed in thefetal liver and adult-derived tissues such as the kidney, bone marrow,and trachea. Besides those tissues, the RNA band was also weaklydetected in the fetal kidney and lung, and adult tissues includingskeletal muscle, lung, placenta, prostate, spleen, and spinal cord. TheRNA expression was also detected in tumor cell lines such as HeLa S3,K562, SW480, and A549, and especially, its very intense expression wasobserved in HeLa S3.

EXAMPLE 4 Cloning of Human OCTN2 CDNA

[0056] Data base search using the entire nucleotide sequence of “humanOCTN1” can detect very similar sequences thereto in several parts of thenucleotide sequence of P1 phage clones (P1H24 clones, GenBank accessionNo. L43407, L43408, L46907, L81773, and L43409) derived from q regionsof human chromosome 5. The parts having similarity with the nucleotidesequence of human OCTN1 are separated by the sequences having nosimilarity to the human OCTN1 sequence. The sequence obtained byconnecting these similar parts with each other with reference to thesequence of human OCTN1 has a high homology over a wide range with humanOCTN1, indicating the presence of OCTN1 homologues. The genomic sequenceregistered in data base was an incomplete one without covering theentire coding region, and, from only this sequence, it was impossible toknow the complete structure of a protein partially encoded by thesequence. Therefore, cDNA cloning of this OCTN1 homologous gene (OCTN2)was performed to determine the coded protein structure. First, 631R S4primer (51-GTGCTGTTGGGCTCCTTCATTTCA-3′, SEQ ID NO: 13) and 631R Alprimer (5′-AGCTGCATGAAGAGAAGGACACTG-3′, SEQ ID NO: 14) were preparedbased on sequences of these P1 phage clones. PCR was performed using aset of these primers and cDNA synthesized from poly(A)+ RNA derived fromthe human adult kidney (CLONTECH) as a template, under the followingconditions: 1 cycle of 94° C. for 3 min; 35 cycles of 94° C. for 30 s,58° C. for 1 min, and 72° C. for 2 min,; and 1 cycle of 72° C. for 10min, resulting in amplification of about 900 bp fragment. This fragmentwas subcloned into the pT7Blue-T vector (Novagen) by the TA cloningmethod-to determine its nucleotide sequence, which clearly showed a veryhigh overall homology with human OCTN1. Therefore, this gene wasdesignated as human OCTN2, and longer cDNAs were cloned.

[0057] The cDNA library derived from the human kidney was screened usingthe cDNA insert of this clone as a probe in the same manner as for humanOCTN1 cDNA cloning, and cDNA containing the entire coding region ofhuman OCTN2 was cloned by a procedure for isolating longer clone and theRapid Amplification of cDNA Ends (RACE) method (Chenchik, A., Moqadam,F., and Siebert, P. (1995), CLONTECHniques X, 5-8), etc. to determineits structure (SEQ ID NO: 4). Specifically, the RACE method was carriedout as follows. The 631R S6 primer (5′-AGCATCCTGTCTCCCTACTTCGTT-3′, SEQID NO: 15) was prepared. PCR was performed using this primer and theMarathon-Ready™ cDNA derived from the human adult kidney (CLONTECH) as atemplate under the following conditions: 94° C. for 2 min; 35 cycles of94° C. for 30 s, 60° C. for 1 min, and 72° C. for 3 min,; and 72° C. for10 min, resulting in amplification of about 1.7 kbp cDNA fragment of the3′-end. This fragment was subcloned into the pT7Blue-T vector by the TAcloning method to determine its structure.

[0058] It became evident that human OCTN2 contains an open reading frame(ORF) encoding a protein consisting of 557 amino acid residues. FIG. 3compares amino acid sequences of human OCTN1 and human OCTN2. Bothshowed overall amino acid homology as high as about 76%. In addition,one consensus sequence (160 to 176) of sugar transporter was present inthe amino acid sequence of human OCTN2 like human OCTN1. These factsindicated that human OCTN2 can be a novel transporter that isstructurally related to human OCTN1. Furthermore, a consensus sequence(218 to 225) of the ATP/GTP binding site was also present in the aminoacid sequence of human OCTN2 like in human OCTN1.

EXAMPLE 5 Northern Analysis

[0059] Northern analysis was performed using about 900 bp human OCTN2cDNA as a probe which was obtained by PCR with a set of 631R S4 primer(5′-GTGCTGTTGGGCTCCTTCATTTCA-3′, SEQ ID NO: 13) and 631R Al primer(5′-AGCTGCATGAAGAGAAGGACACTG-3′, SEQ ID NO: 14) in the same manner asfor human OCTN1. The results are shown in FIG. 4. Although theexpression pattern of human OCTN2 partly overlapped with that of humanOCTN1, human OCTN2 differs from human OCTN1 in that the former was veryintensely expressed in the kidney among fetal tissues, while the latterwas strongly expressed also in cancer cell strains such as K-562, HeLaS3, SW480, etc. as well as the kidney, indicating that OCTN1 and OCTN2may be involved in transport of substances such as carcinostatics inthese cancer cells.

EXAMPLE 6 Forced Expression of Human OCTN1 in Human Fetal Kidney Cells(HEK293) and its Activity Determination

[0060] Phage DNAs were extracted from positive phage clones obtained byscreening the clones by the plaque hybridization method using the QIAGENLambda Kit (QIAGEN). After the DNA insert was subcloned into the pUC18vector, cDNA containing the entire ORF which was cleaved out with SmaIand EcoRI was integrated between the EcoRI site and the blunted HindIIIsite of an expression vector for mammalian cells, pcDNA3 (Invitrogen),to obtain an expression plasmid DNA, pcDNA3/OCTN1. Plasmid DNA wasprepared by alkaline-SDS method using the QIAGEN PLASMID MAXI Kit(QIAGEN).

[0061] The human fetal-kidney-derived cell strain, HEK 293 cells weretransfected with the plasmid pcDNA3/OCTN1 and pcDNA3 vector containingno insert as a control by the calcium phosphate method. First, theplasmid DNA (10 μg), a Hepes buffer solution (137 mM NaCl, 5 mM KCl, 0.7mM Na₂HPO₄, 6 mM Dextrose, and 21 mM Hepes pH 7.1) (1 ml), and 2 M CaCl₂(62.5 μl) were combined and allowed to stand at room temperature for 30min or more to form calcium phosphate coprecipitates. After cells wereplated on 10-cm plates at 1.5×10⁶ cells per plate and cultured for 24 h,the calcium phosphate coprecipitates were added thereto, and the cellswere further cultured for 24 h. Then, plates were washed with phosphatebuffered saline (PBS), and the cells were further cultured for 24 hafter the addition of fresh culture medium.

[0062] Transport experiment was performed using cells transfected withthe plasmid DNA or untreated cells according to the followingprocedures. Cells were detached from plates using a rubber policeman,suspended in a transport buffer (containing 125 mM NaCl, 4.8 mM KCl, 5.6mM (+)-glucose, 1.2 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, and 25 mMHepes pH 7.4), and pre-incubated for 20 min. An appropriate amount ofeach labeled substrate ([¹⁴C]TEA (tetraethylammonium) (NEN),[³H]carnitine (L-carnitine hydrochloride) (Amersham), [³H]PCG(benzylpenicillin) (Amersham), [³H]quinidine (ARC), or [³H]pyrilamine(mepyramine) (Amersham)) was then added to the cell suspension, and theresulting mixture was incubated at 37° C. for a predetermined period oftime. Incubated cells were overlaid on a silicon layer prepared bylayering a mixture of silicon oil and liquid paraffin (specific gravity1.022) on a 3 M KCl layer, and separated by centrifugation.Radioactivity of cells was measured to determine the into-the-celltransport activity. In this case, 1×10⁶ cells were used as one point ofcells. HEK 293 cells were cultured in Dulbecco's MEM containing 10%fetal calf serum (FCS) in an atmosphere of 5% carbon dioxide at 37° C.

[0063] First, the transporter capacity was measured in the cellstransfected with pcDNA3/OCTN1 and untreated cells using TEA as asubstrate (FIG. 5). A reaction time-dependent TEA uptake into the humanOCTN1-transfectd cells was clearly observed. This uptake was notobserved in untreated cells. Next, effects of the addition of unlabeledTEA on the labeled substrate uptake in this system (cold inhibition) wasexamined (FIG. 6). A decrease in the apparent uptake of the labeledsubstrate was clearly seen depending on the concentration of cold TEAadded. In this experiment, almost no uptake of the substrate into cellswas observed in cells transfected with the pcDNA3 vector containing noinsert (Mock) used as a control like in untreated cells used, clearlyindicating that this uptake phenomenon is due to the transfection of thecells with human OCTN1. Next, to obtain the Km (Michaelis constant)value of human OCTNL to TEA, the uptake of ¹⁴C-TEA with variousconcentrations was measured (FIG. 7). From Lineweaver-Burk reciprocalplot of the net uptake obtained by subtracting the amount of the uptakein Mock cells from that in the human OCTN1-transfected cells, the Kmvalue of 0.44±0.04 mM was obtained with the maximal velocity, Vmax of6.68±0.34 (nmol/3 min/mg). Next, the transport capacity of human OCTN1for other substrate than TEA was examined (FIG. 8). When the transportcapacity was measured using labeled organic cations such as labeledcarnitine, quinidine, and pyrilamine, a significant increase in theuptake of these compounds was clearly observed in humanOCTN1-transfected cells as compared with Mock cells, clearly indicatingthat these organic cations can serve as substrates for human OCTN1.However, no significant increase in the uptake of an organic anion, PCG(benzylpenicillin), was observed.

EXAMPLE 7 Activity Measurement Of Human OCTN1 Using Xenopus Oocytes

[0064] cRNA was synthesized in vitro using T7 RNA polymerase withpcDNA3/OCTN1 as a template. This cRNA was diluted to the concentrationof 0.3 ng/nl, and its 50-nl (15 ng) aliquot was injected into a singleoocyte. As a control, 50 nl of distilled water was injected. Theseoocytes were cultured for 3 days, and then used for the transportexperiment. After being preincubated in an uptake buffer (0.05% Tween80, 100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM Hepes pH7.4) at 25° C. for 20 min, the oocytes were transferred to the uptakebuffer containing 0.5 ml of labeled substrate to initiate the uptake.After the incubation at 25° C. for 1 h, the oocytes were washed in theice-cold uptake buffer three times to terminate the reaction. Theoocytes were solubilized in 5% SDS and mixed with Cleasol I (a cocktailfor liquid scintillation counter) (3 ml) to determine the radioactivity.The radioactivity of the uptake buffer which contained the labeledcompound at the time of incubation (external solution) (10 μl) was alsosimilarly measured. The ratio of the radioactivity (dpm value) in theoocytes to that (dpm value) in the external solution was used as theuptake activity.

[0065] Human OCTN1 also expresses the transport capacity for organiccations such as quinidine, mepyramine and carnitine, as well as TEA inthis transport experiment system using Xenopus oocytes (FIG. 9).

[0066] Next, the transport capacity of human OCTN1 for carcinostatics,etc. was examined. The results revealed that human OCTN1 has theactivity to transport actinomycin D, etoposide, vinblastine, anddaunomycin (FIG. 10). These results strongly indicate that OCTN1 wouldbe involved in the into-the-cell translocation mechanism (mechanism forabsorption by cells) for these drugs, which have been clinically used ascarcinostatics. By designing and screening drugs utilizing the substratespecificity of OCTN1 so as to be readily recognized by this transporter,it would be possible to efficiently develop useful drugs that can bereadily absorbed by the cells.

EXAMPLE 8 Forced expression of human OCTN2 in HEK cells and its activitymeasurement

[0067] The expression plasmid DNA for human OCTN2 in mammalian cells wasprepared as follows.

[0068] A single-stranded cDNA was synthesized from poly(A)+ RNA derivedfrom the human fetal kidney (CLONTECH) using the SuperScript™ II reversetranscriptase (GIBCO BRL). PCR was performed using the thus-obtainedcDNA as a template under the following conditions to amplify 5′- and3′-end fragments of human OCTN2.

[0069] For the amplification of 5′-end fragment (about 800 bp) of humanOCTN2, OCTN2 3 primer (5′-GATGGATCCCGGACGGTCTTGGGTCGCCTGCTG-3′, SEQ IDNO: 16) and OCN2 4 primer (5′-GATGGATCCAAATGCTGCCACATAGTTGGAGAT-3′, SEQID NO: 17) were used. PCR was carried out using DNA polymerase ExTaq(TaKaRa) and dNTPs (150 μM 7-deaza dGTP, 50 μM dGTP, 200 μM DATP, 200 μMdTTP, and 200 μM dCTP) according to the following conditions:94° C. for2 min; 35 cycles of 94° C. for 30 s, 63° C. for 1 min, and 72° C. for 2min,; and 72° C. for 10 min. For the amplification of 3′-end fragment(about 1.2 kbp) of human OCTN2, OCTN2 7 primer(5′-GATGGATCCATGGGCATGCAGACAGGCTTCAGC-3′, SEQ ID NO: 18) and OCTN2 8primer (5′-GATGGATCCTTCCTCTTCAGTTTCTCCCTTACT-3′, SEQ ID NO: 19) wereused. PCR was carried out using DNA polymerase ExTaq (TaKaRa) and dNTPs(200 μM dGTP, 200 μM DATP, 200 μM dTTP, and 200 μM dCTP) according tothe following conditions:94° C. for 2 min; 35 cycles of 94° C. for 30 s,63° C. for 30 s, and 72° C. for 2 min,; and 72° C. for 10 min.

[0070] These fragments were respectively electrophoresed on agarose gel,excised from the gel, purified, and subcloned into the pT7Blue-T vector.Clones having no PCR error were selected by sequencing, and clones fromboth fragments were ligated at the PstI site in the overlapping region.Each ligated fragment was eventually incorporated into the BamHI site ofthe pcDNA3 vector, and used as the expression plasmid DNA pcDNA3/OCTN2.

[0071] HEK cells were transfected with pcDNA3/OCTN2, the pcDNA3 vectorcontaining no insert (Mock), or pcDNA3/OCTN1 by the method described inExample 6 to perform transport experiments. It was proved that humanOCTN2 has a high capacity to efficiently transport carnitine (FIG. 11).On the other hand, human OCTN2 hardly transported TEA, which wereefficiently transported by human OCTN1, revealing that they clearlydiffer in their substrate specificities.

[0072] Next, Na⁺ dependence of human OCTN2-mediated carnitine transportwas examined using a transport buffer in which Na⁺ was replaced withK+(FIG. 12). The result showed that carnitine transport mediated byhuman OCTN2 completely depended on the presence of Na⁺, indicating thatOCTN2 is a symport type transporter that transports substrates and Na⁺in the same direction.

EXAMPLE 9 Cloning of mouse OCTN1

[0073] Data base search using human OCTN1 cDNA sequence detected severalExpressed Sequence Tags (ESTs) derived from mouse, which had very highhomology to the human OCTN1 cDNA sequence. Based on these EST sequences,MONL 1 primer (5′-CGCGCCGAATCGCTGAATCCTTTC-3′, SEQ ID NO: 20) and MONA 4primer (5′-AGGCTTTTGATTTGTTCTGTTGAG-3′, SEQ ID NO: 21) were prepared.PCR was performed using a set of these primers and cDNA prepared frompoly(A)⁺ RNA derived from the mouse kidney as a template. As a result,fragments of about 2 kbp were amplified. These fragments wereelectrophoresed on agarose gels, excised from the gels, purified, andsubcloned into the pT7Blue T vector (Novagen) by the TA cloning method.The sequence of mouse OCTN1 was determined by sequencing plural clones.The nucleotide sequence of cDNA thus determined is shown in SEQ ID NO:23, and amino acid sequence of the protein encoded by the cDNA in SEQ IDNO: 22.

EXAMPLE 10 Cloning of Mouse OCTN2

[0074] First, MONB 20 primer (5′-CCCATGCCAACAAGGACAAAAAGC-3′, SEQ ID NO:24) was prepared from the sequence of human OCTN2 cDNA. TheMarathon-Ready™ cDNA derived from the mouse kidney (CLONTECH) was usedas a template for the 5′-Rapid Amplification of cDNA ends (RACE) toclone the 5′-end sequence upstream of the primer. Next, data base searchwas performed using human OCTN2 nucleotide sequence to detect severalESTs derived from mouse, which had a very high homology with humanOCTN2. MONB 26 primer (5′-ACAGAACAGAAAAGCCCTCAGTCA-3′, SEQ ID NO: 25)was prepared from these EST sequences. MONB 6 primer(5′-TGTTTTTCGTGGGTGTGCTGATGG-3′, SEQ ID NO: 26) was prepared from thesequence obtained by the 5′-RACE. PCR was performed using this primerand MONB 26 primer and cDNA prepared from poly(A)+ RNA derived from themouse kidney as a template to amplify the 3′-end fragments. The sequenceof mouse OCTN2 was determined by sequencing directly of after subcloningrespective fragments. The nucleotide sequence of the cDNA thusdetermined is shown in SEQ ID NO: 28, and amino acid sequence of theprotein encoded by the cDNA in SEQ ID NO: 27.

EXAMPLE 11 Tissue Expression Analysis of Mouse OCTN1 and MOUSE OCTN2

[0075] The expression amount of mouse OCTNL and mouse OCTN2 genes invarious tissues was examined by RT-PCR using a mouse Multiple TissuecDNA (MTC) panel (CLONTECH) (FIG. 13). Primers used are MONL 1 and MONA4 for mouse OCTN1, and MONB 6 and MONB 26 for mouse OCTN2. As a result,the high level expression of mouse OCTN1 was detected in the kidney andliver, while that of mouse OCTN2 in the kidney, liver, and 7-days oldembryo.

INDUSTRIAL APPLICABILITY

[0076] This invention provides a family of novel organic cationtransporter genes and proteins-encoded by these genes. Transporterproteins of this invention are useful for developing newly designeddrugs that can be transported mediated by these proteins, andpharmaceuticals for disorders caused by functional abnormalities of theproteins.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 33 <210> SEQ ID NO 1<211> LENGTH: 551 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 1 Met Arg Asp Tyr Asp Glu Val Ile Ala Phe Leu Gly Glu Trp GlyPro 1 5 10 15 Phe Gln Arg Leu Ile Phe Phe Leu Leu Ser Ala Ser Ile IlePro Asn 20 25 30 Gly Phe Asn Gly Met Ser Val Val Phe Leu Ala Gly Thr ProGlu His 35 40 45 Arg Cys Arg Val Pro Asp Ala Ala Asn Leu Ser Ser Ala TrpArg Asn 50 55 60 Asn Ser Val Pro Leu Arg Leu Arg Asp Gly Arg Glu Val ProHis Ser 65 70 75 80 Cys Ser Arg Tyr Arg Leu Ala Thr Ile Ala Asn Phe SerAla Leu Gly 85 90 95 Leu Glu Pro Gly Arg Asp Val Asp Leu Gly Gln Leu GluGln Glu Ser 100 105 110 Cys Leu Asp Gly Trp Glu Phe Ser Gln Asp Val TyrLeu Ser Thr Val 115 120 125 Val Thr Glu Trp Asn Leu Val Cys Glu Asp AsnTrp Lys Val Pro Leu 130 135 140 Thr Thr Ser Leu Phe Phe Val Gly Val LeuLeu Gly Ser Phe Val Ser 145 150 155 160 Gly Gln Leu Ser Asp Arg Phe GlyArg Lys Asn Val Leu Phe Ala Thr 165 170 175 Met Ala Val Gln Thr Gly PheSer Phe Leu Gln Ile Phe Ser Ile Ser 180 185 190 Trp Glu Met Phe Thr ValLeu Phe Val Ile Val Gly Met Gly Gln Ile 195 200 205 Ser Asn Tyr Val ValAla Phe Ile Leu Gly Thr Glu Ile Leu Gly Lys 210 215 220 Ser Val Arg IleIle Phe Ser Thr Leu Gly Val Cys Thr Phe Phe Ala 225 230 235 240 Val GlyTyr Met Leu Leu Pro Leu Phe Ala Tyr Phe Ile Arg Asp Trp 245 250 255 ArgMet Leu Leu Leu Ala Leu Thr Val Pro Gly Val Leu Cys Val Pro 260 265 270Leu Trp Trp Phe Ile Pro Glu Ser Pro Arg Trp Leu Ile Ser Gln Arg 275 280285 Arg Phe Arg Glu Ala Glu Asp Ile Ile Gln Lys Ala Ala Lys Met Asn 290295 300 Asn Thr Ala Val Pro Ala Val Ile Phe Asp Ser Val Glu Glu Leu Asn305 310 315 320 Pro Leu Lys Gln Gln Lys Ala Phe Ile Leu Asp Leu Phe ArgThr Arg 325 330 335 Asn Ile Ala Ile Met Thr Ile Met Ser Leu Leu Leu TrpMet Leu Thr 340 345 350 Ser Val Gly Tyr Phe Ala Leu Ser Leu Asp Ala ProAsn Leu His Gly 355 360 365 Asp Ala Tyr Leu Asn Cys Phe Leu Ser Ala LeuIle Glu Ile Pro Ala 370 375 380 Tyr Ile Thr Ala Trp Leu Leu Leu Arg ThrLeu Pro Arg Arg Tyr Ile 385 390 395 400 Ile Ala Ala Val Leu Phe Trp GlyGly Gly Val Leu Leu Phe Ile Gln 405 410 415 Leu Val Pro Val Asp Tyr TyrPhe Leu Ser Ile Gly Leu Val Met Leu 420 425 430 Gly Lys Phe Gly Ile ThrSer Ala Phe Ser Met Leu Tyr Val Phe Thr 435 440 445 Ala Glu Leu Tyr ProThr Leu Val Arg Asn Met Ala Val Gly Val Thr 450 455 460 Ser Thr Ala SerArg Val Gly Ser Ile Ile Ala Pro Tyr Phe Val Tyr 465 470 475 480 Leu GlyAla Tyr Asn Arg Met Leu Pro Tyr Ile Val Met Gly Ser Leu 485 490 495 ThrVal Leu Ile Gly Ile Phe Thr Leu Phe Phe Pro Glu Ser Leu Gly 500 505 510Met Thr Leu Pro Glu Thr Leu Glu Gln Met Gln Lys Val Lys Trp Phe 515 520525 Arg Ser Gly Lys Lys Thr Arg Asp Ser Met Glu Thr Glu Glu Asn Pro 530535 540 Lys Val Leu Ile Thr Ala Phe 545 550 <210> SEQ ID NO 2 <211>LENGTH: 2135 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (147)..(1799) <400> SEQUENCE: 2ccccggcttc gcgccccaat ttctaacagc ctgcctgtcc cccgggaacg ttctaacatc 60cttggggagc gccccagcta caagacactg tcctgagaac gctgtcatca cccgtagttg 120caagtttcgg agcggcagtg ggaagc atg cgg gac tac gac gag gtg atc gcc 173 MetArg Asp Tyr Asp Glu Val Ile Ala 1 5 ttc ctg ggc gag tgg ggg ccc ttc cagcgc ctc atc ttc ttc ctg ctc 221 Phe Leu Gly Glu Trp Gly Pro Phe Gln ArgLeu Ile Phe Phe Leu Leu 10 15 20 25 agc gcc agc atc atc ccc aat ggc ttcaat ggt atg tca gtc gtg ttc 269 Ser Ala Ser Ile Ile Pro Asn Gly Phe AsnGly Met Ser Val Val Phe 30 35 40 ctg gcg ggg acc ccg gag cac cgc tgt cgagtg ccg gac gcc gcg aac 317 Leu Ala Gly Thr Pro Glu His Arg Cys Arg ValPro Asp Ala Ala Asn 45 50 55 ctg agc agc gcc tgg cgc aac aac agt gtc ccgctg cgg ctg cgg gac 365 Leu Ser Ser Ala Trp Arg Asn Asn Ser Val Pro LeuArg Leu Arg Asp 60 65 70 ggc cgc gag gtg ccc cac agc tgc agc cgc tac cggctc gcc acc atc 413 Gly Arg Glu Val Pro His Ser Cys Ser Arg Tyr Arg LeuAla Thr Ile 75 80 85 gcc aac ttc tcg gcg ctc ggg ctg gag ccg ggg cgc gacgtg gac ctg 461 Ala Asn Phe Ser Ala Leu Gly Leu Glu Pro Gly Arg Asp ValAsp Leu 90 95 100 105 ggg cag ctg gag cag gag agc tgc ctg gat ggc tgggag ttc agc cag 509 Gly Gln Leu Glu Gln Glu Ser Cys Leu Asp Gly Trp GluPhe Ser Gln 110 115 120 gac gtc tac ctg tcc acc gtc gtg acc gag tgg aatctg gtg tgt gag 557 Asp Val Tyr Leu Ser Thr Val Val Thr Glu Trp Asn LeuVal Cys Glu 125 130 135 gac aac tgg aag gtg ccc ctc acc acc tcc ctg ttcttc gta ggc gtg 605 Asp Asn Trp Lys Val Pro Leu Thr Thr Ser Leu Phe PheVal Gly Val 140 145 150 ctc ctc ggc tcc ttc gtg tcc ggg cag ctg tca gacagg ttt ggc agg 653 Leu Leu Gly Ser Phe Val Ser Gly Gln Leu Ser Asp ArgPhe Gly Arg 155 160 165 aag aac gtt ctc ttc gca acc atg gct gta cag actggc ttc agc ttc 701 Lys Asn Val Leu Phe Ala Thr Met Ala Val Gln Thr GlyPhe Ser Phe 170 175 180 185 ctg cag att ttc tcc atc agc tgg gag atg ttcact gtg tta ttt gtc 749 Leu Gln Ile Phe Ser Ile Ser Trp Glu Met Phe ThrVal Leu Phe Val 190 195 200 atc gtg ggc atg ggc cag atc tcc aac tat gtggta gcc ttc ata cta 797 Ile Val Gly Met Gly Gln Ile Ser Asn Tyr Val ValAla Phe Ile Leu 205 210 215 gga aca gaa att ctt ggc aag tca gtt cgt attata ttc tct aca tta 845 Gly Thr Glu Ile Leu Gly Lys Ser Val Arg Ile IlePhe Ser Thr Leu 220 225 230 gga gtg tgc aca ttt ttt gca gtt ggc tat atgctg ctg cca ctg ttt 893 Gly Val Cys Thr Phe Phe Ala Val Gly Tyr Met LeuLeu Pro Leu Phe 235 240 245 gct tac ttc atc aga gac tgg cgg atg ctg ctgctg gcg ctg acg gtg 941 Ala Tyr Phe Ile Arg Asp Trp Arg Met Leu Leu LeuAla Leu Thr Val 250 255 260 265 ccg gga gtg ctg tgt gtc ccg ctg tgg tggttc att cct gaa tct ccc 989 Pro Gly Val Leu Cys Val Pro Leu Trp Trp PheIle Pro Glu Ser Pro 270 275 280 cga tgg ctg ata tcc cag aga aga ttt agagag gct gaa gat atc atc 1037 Arg Trp Leu Ile Ser Gln Arg Arg Phe Arg GluAla Glu Asp Ile Ile 285 290 295 caa aaa gct gca aaa atg aac aac aca gctgta cca gca gtg ata ttt 1085 Gln Lys Ala Ala Lys Met Asn Asn Thr Ala ValPro Ala Val Ile Phe 300 305 310 gat tct gtg gag gag cta aat ccc ctg aagcag cag aaa gct ttc att 1133 Asp Ser Val Glu Glu Leu Asn Pro Leu Lys GlnGln Lys Ala Phe Ile 315 320 325 ctg gac ctg ttc agg act cgg aat att gccata atg acc att atg tct 1181 Leu Asp Leu Phe Arg Thr Arg Asn Ile Ala IleMet Thr Ile Met Ser 330 335 340 345 ttg ctg cta tgg atg ctg acc tca gtgggt tac ttt gct ctg tct ctg 1229 Leu Leu Leu Trp Met Leu Thr Ser Val GlyTyr Phe Ala Leu Ser Leu 350 355 360 gat gct cct aat tta cat gga gat gcctac ctg aac tgt ttc ctc tct 1277 Asp Ala Pro Asn Leu His Gly Asp Ala TyrLeu Asn Cys Phe Leu Ser 365 370 375 gcc ttg att gaa att cca gct tac attaca gcc tgg ctg cta ttg cga 1325 Ala Leu Ile Glu Ile Pro Ala Tyr Ile ThrAla Trp Leu Leu Leu Arg 380 385 390 acg ctg ccc agg cgt tat atc ata gctgca gta ctg ttc tgg gga gga 1373 Thr Leu Pro Arg Arg Tyr Ile Ile Ala AlaVal Leu Phe Trp Gly Gly 395 400 405 ggt gtg ctt ctc ttc att caa ctg gtacct gtg gat tat tac ttc tta 1421 Gly Val Leu Leu Phe Ile Gln Leu Val ProVal Asp Tyr Tyr Phe Leu 410 415 420 425 tcc att ggt ctg gtc atg ctg ggaaaa ttt ggg atc acc tct gct ttc 1469 Ser Ile Gly Leu Val Met Leu Gly LysPhe Gly Ile Thr Ser Ala Phe 430 435 440 tcc atg ctg tat gtc ttc act gctgag ctc tac cca acc ctg gtc agg 1517 Ser Met Leu Tyr Val Phe Thr Ala GluLeu Tyr Pro Thr Leu Val Arg 445 450 455 aac atg gcg gtg ggg gtc aca tccacg gcc tcc aga gtg ggc agc atc 1565 Asn Met Ala Val Gly Val Thr Ser ThrAla Ser Arg Val Gly Ser Ile 460 465 470 att gcc ccc tac ttt gtt tac ctcggt gct tac aac aga atg ctg ccc 1613 Ile Ala Pro Tyr Phe Val Tyr Leu GlyAla Tyr Asn Arg Met Leu Pro 475 480 485 tac atc gtc atg ggt agt ctg actgtc ctg att gga atc ttc acc ctt 1661 Tyr Ile Val Met Gly Ser Leu Thr ValLeu Ile Gly Ile Phe Thr Leu 490 495 500 505 ttt ttc cct gaa agt ttg ggaatg act ctt cca gaa acc tta gag cag 1709 Phe Phe Pro Glu Ser Leu Gly MetThr Leu Pro Glu Thr Leu Glu Gln 510 515 520 atg cag aaa gtg aaa tgg ttcaga tct ggg aaa aaa aca aga gac tca 1757 Met Gln Lys Val Lys Trp Phe ArgSer Gly Lys Lys Thr Arg Asp Ser 525 530 535 atg gag aca gaa gaa aat cccaag gtt cta ata act gca ttc 1799 Met Glu Thr Glu Glu Asn Pro Lys Val LeuIle Thr Ala Phe 540 545 550 tgaaaaaata tctaccccat ttggtgaagt gaaaaacagaaaaataagac cctgtggaga 1859 aattcgttgt tcccactgaa atggactgac tgtaacgattgacaccaaaa tgaaccttgc 1919 tatcaagaaa tgctcgtcat acagtaaact ctggatgattcttccagata atgtccttgc 1979 tttacaaacc aaccatttct agagagtctc cttactcattaattcaatga aatggattgg 2039 taagatgtct tgaaaacatg ttagtcaagg actggtaaaatacatataaa gattaacact 2099 catttccaat catacaaata ctatccaaat aaaaat 2135<210> SEQ ID NO 3 <211> LENGTH: 557 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 3 Met Arg Asp Tyr Asp Glu Val Thr Ala Phe LeuGly Glu Trp Gly Pro 1 5 10 15 Phe Gln Arg Leu Ile Phe Phe Leu Leu SerAla Ser Ile Ile Pro Asn 20 25 30 Gly Phe Thr Gly Leu Ser Ser Val Phe LeuIle Ala Thr Pro Glu His 35 40 45 Arg Cys Arg Val Pro Asp Ala Ala Asn LeuSer Ser Ala Trp Arg Asn 50 55 60 His Thr Val Pro Leu Arg Leu Arg Asp GlyArg Glu Val Pro His Ser 65 70 75 80 Cys Arg Arg Tyr Arg Leu Ala Thr IleAla Asn Phe Ser Ala Leu Gly 85 90 95 Leu Glu Pro Gly Arg Asp Val Asp LeuGly Gln Leu Glu Gln Glu Ser 100 105 110 Cys Leu Asp Gly Trp Glu Phe SerGln Asp Val Tyr Leu Ser Thr Ile 115 120 125 Val Thr Glu Trp Asn Leu ValCys Glu Asp Asp Trp Lys Ala Pro Leu 130 135 140 Thr Ile Ser Leu Phe PheVal Gly Val Leu Leu Gly Ser Phe Ile Ser 145 150 155 160 Gly Gln Leu SerAsp Arg Phe Gly Arg Lys Asn Val Leu Phe Val Thr 165 170 175 Met Gly MetGln Thr Gly Phe Ser Phe Leu Gln Ile Phe Ser Lys Asn 180 185 190 Phe GluMet Phe Val Val Leu Phe Val Leu Val Gly Met Gly Gln Ile 195 200 205 SerAsn Tyr Val Ala Ala Phe Val Leu Gly Thr Glu Ile Leu Gly Lys 210 215 220Ser Val Arg Ile Ile Phe Ser Thr Leu Gly Val Cys Ile Phe Tyr Ala 225 230235 240 Phe Gly Tyr Met Val Leu Pro Leu Phe Ala Tyr Phe Ile Arg Asp Trp245 250 255 Arg Met Leu Leu Val Ala Leu Thr Met Pro Gly Val Leu Cys ValAla 260 265 270 Leu Trp Trp Phe Ile Pro Glu Ser Pro Arg Trp Leu Ile SerGln Gly 275 280 285 Arg Phe Glu Glu Ala Glu Val Ile Ile Arg Lys Ala AlaLys Ala Asn 290 295 300 Gly Ile Val Val Pro Ser Thr Ile Phe Asp Pro SerGlu Leu Gln Asp 305 310 315 320 Leu Ser Ser Lys Lys Gln Gln Ser His AsnIle Leu Asp Leu Leu Arg 325 330 335 Thr Trp Asn Ile Arg Met Val Thr IleMet Ser Ile Met Leu Trp Met 340 345 350 Thr Ile Ser Val Gly Tyr Phe GlyLeu Ser Leu Asp Thr Pro Asn Leu 355 360 365 His Gly Asp Ile Phe Val AsnCys Phe Leu Ser Ala Met Val Glu Val 370 375 380 Pro Ala Tyr Val Leu AlaTrp Leu Leu Leu Gln Tyr Leu Pro Arg Arg 385 390 395 400 Tyr Ser Met AlaThr Ala Leu Phe Leu Gly Gly Ser Val Leu Leu Phe 405 410 415 Met Gln LeuVal Pro Pro Asp Leu Tyr Tyr Leu Ala Thr Val Leu Val 420 425 430 Met ValGly Lys Phe Gly Val Thr Ala Ala Phe Ser Met Val Tyr Val 435 440 445 TyrThr Ala Glu Leu Tyr Pro Thr Val Val Arg Asn Met Gly Val Gly 450 455 460Val Ser Ser Thr Ala Ser Arg Leu Gly Ser Ile Leu Ser Pro Tyr Phe 465 470475 480 Val Tyr Leu Gly Ala Tyr Asp Arg Phe Leu Pro Tyr Ile Leu Met Gly485 490 495 Ser Leu Thr Ile Leu Thr Ala Ile Leu Thr Leu Phe Leu Pro GluSer 500 505 510 Phe Gly Thr Pro Leu Pro Asp Thr Ile Asp Gln Met Leu ArgVal Lys 515 520 525 Gly Met Lys His Arg Lys Thr Pro Ser His Thr Arg MetLeu Lys Asp 530 535 540 Gly Gln Glu Arg Pro Thr Ile Leu Lys Ser Thr AlaPhe 545 550 555 <210> SEQ ID NO 4 <211> LENGTH: 1831 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (124)..(1794) <400> SEQUENCE: 4 cggacggtct tgggtcgcctgctgcctggc ttgcctggtc ggcggcgggt gccccgcgcg 60 cacgcgcaaa gcccgccgcgttcccagacc ccaggccgcg ctctgtgggc ctctgagggc 120 ggc atg cgg gac tac gacgag gtg acc gcc ttc ctg ggc gag tgg ggg 168 Met Arg Asp Tyr Asp Glu ValThr Ala Phe Leu Gly Glu Trp Gly 1 5 10 15 ccc ttc cag cgc ctc atc ttcttc ctg ctc agc gcc agc atc atc ccc 216 Pro Phe Gln Arg Leu Ile Phe PheLeu Leu Ser Ala Ser Ile Ile Pro 20 25 30 aat ggc ttc acc ggc ctg tcc tccgtg ttc ctg ata gcg acc ccg gag 264 Asn Gly Phe Thr Gly Leu Ser Ser ValPhe Leu Ile Ala Thr Pro Glu 35 40 45 cac cgc tgc cgg gtg ccg gac gcc gcgaac ctg agc agc gcc tgg cgc 312 His Arg Cys Arg Val Pro Asp Ala Ala AsnLeu Ser Ser Ala Trp Arg 50 55 60 aac cac act gtc cca ctg cgg ctg cgg gacggc cgc gag gtg ccc cac 360 Asn His Thr Val Pro Leu Arg Leu Arg Asp GlyArg Glu Val Pro His 65 70 75 agc tgc cgc cgc tac cgg ctc gcc acc atc gccaac ttc tcg gcg ctc 408 Ser Cys Arg Arg Tyr Arg Leu Ala Thr Ile Ala AsnPhe Ser Ala Leu 80 85 90 95 ggg ctg gag ccg ggg cgc gac gtg gac ctg gggcag ctg gag cag gag 456 Gly Leu Glu Pro Gly Arg Asp Val Asp Leu Gly GlnLeu Glu Gln Glu 100 105 110 agc tgt ctg gat ggc tgg gag ttc agt cag gacgtc tac ctg tcc acc 504 Ser Cys Leu Asp Gly Trp Glu Phe Ser Gln Asp ValTyr Leu Ser Thr 115 120 125 att gtg acc gag tgg aac ctg gtg tgt gag gacgac tgg aag gcc cca 552 Ile Val Thr Glu Trp Asn Leu Val Cys Glu Asp AspTrp Lys Ala Pro 130 135 140 ctc aca atc tcc ttg ttc ttc gtg ggt gtg ctgttg ggc tcc ttc att 600 Leu Thr Ile Ser Leu Phe Phe Val Gly Val Leu LeuGly Ser Phe Ile 145 150 155 tca ggg cag ctg tca gac agg ttt ggc cgg aagaat gtg ctg ttc gtg 648 Ser Gly Gln Leu Ser Asp Arg Phe Gly Arg Lys AsnVal Leu Phe Val 160 165 170 175 acc atg ggc atg cag aca ggc ttc agc ttcctg cag atc ttc tcg aag 696 Thr Met Gly Met Gln Thr Gly Phe Ser Phe LeuGln Ile Phe Ser Lys 180 185 190 aat ttt gag atg ttt gtc gtg ctg ttt gtcctt gta ggc atg ggc cag 744 Asn Phe Glu Met Phe Val Val Leu Phe Val LeuVal Gly Met Gly Gln 195 200 205 atc tcc aac tat gtg gca gca ttt gtc ctgggg aca gaa att ctt ggc 792 Ile Ser Asn Tyr Val Ala Ala Phe Val Leu GlyThr Glu Ile Leu Gly 210 215 220 aag tca gtt cgt ata ata ttc tct acg ttagga gtg tgc ata ttt tat 840 Lys Ser Val Arg Ile Ile Phe Ser Thr Leu GlyVal Cys Ile Phe Tyr 225 230 235 gca ttt ggc tac atg gtg ctg cca ctg tttgct tac ttc atc cga gac 888 Ala Phe Gly Tyr Met Val Leu Pro Leu Phe AlaTyr Phe Ile Arg Asp 240 245 250 255 tgg cgg atg ctg ctg gtg gcg ctg acgatg ccg ggg gtg ctg tgc gtg 936 Trp Arg Met Leu Leu Val Ala Leu Thr MetPro Gly Val Leu Cys Val 260 265 270 gca ctc tgg tgg ttc atc cct gag tccccc cga tgg ctc atc tct cag 984 Ala Leu Trp Trp Phe Ile Pro Glu Ser ProArg Trp Leu Ile Ser Gln 275 280 285 gga cga ttt gaa gag gca gag gtg atcatc cgc aag gct gcc aaa gcc 1032 Gly Arg Phe Glu Glu Ala Glu Val Ile IleArg Lys Ala Ala Lys Ala 290 295 300 aat ggg att gtt gtg cct tcc act atcttt gac ccg agt gag tta caa 1080 Asn Gly Ile Val Val Pro Ser Thr Ile PheAsp Pro Ser Glu Leu Gln 305 310 315 gac cta agt tcc aag aag cag cag tcccac aac att ctg gat ctg ctt 1128 Asp Leu Ser Ser Lys Lys Gln Gln Ser HisAsn Ile Leu Asp Leu Leu 320 325 330 335 cga acc tgg aat atc cgg atg gtcacc atc atg tcc ata atg ctg tgg 1176 Arg Thr Trp Asn Ile Arg Met Val ThrIle Met Ser Ile Met Leu Trp 340 345 350 atg acc ata tca gtg ggc tat tttggg ctt tcg ctt gat act cct aac 1224 Met Thr Ile Ser Val Gly Tyr Phe GlyLeu Ser Leu Asp Thr Pro Asn 355 360 365 ttg cat ggg gac atc ttt gtg aactgc ttc ctt tca gcg atg gtt gaa 1272 Leu His Gly Asp Ile Phe Val Asn CysPhe Leu Ser Ala Met Val Glu 370 375 380 gtc cca gca tat gtg ttg gcc tggctg ctg ctg caa tat ttg ccc cgg 1320 Val Pro Ala Tyr Val Leu Ala Trp LeuLeu Leu Gln Tyr Leu Pro Arg 385 390 395 cgc tat tcc atg gcc act gcc ctcttc ctg ggt ggc agt gtc ctt ctc 1368 Arg Tyr Ser Met Ala Thr Ala Leu PheLeu Gly Gly Ser Val Leu Leu 400 405 410 415 ttc atg cag ctg gta ccc ccagac ttg tat tat ttg gct aca gtc ctg 1416 Phe Met Gln Leu Val Pro Pro AspLeu Tyr Tyr Leu Ala Thr Val Leu 420 425 430 gtg atg gtg ggc aag ttt ggagtc acg gct gcc ttt tcc atg gtc tac 1464 Val Met Val Gly Lys Phe Gly ValThr Ala Ala Phe Ser Met Val Tyr 435 440 445 gtg tac aca gcc gag ctg tatccc aca gtg gtg aga aac atg ggt gtg 1512 Val Tyr Thr Ala Glu Leu Tyr ProThr Val Val Arg Asn Met Gly Val 450 455 460 gga gtc agc tcc aca gca tcccgc ctg ggc agc atc ctg tct ccc tac 1560 Gly Val Ser Ser Thr Ala Ser ArgLeu Gly Ser Ile Leu Ser Pro Tyr 465 470 475 ttc gtt tac ctt ggt gcc tacgac cgc ttc ctg ccc tac att ctc atg 1608 Phe Val Tyr Leu Gly Ala Tyr AspArg Phe Leu Pro Tyr Ile Leu Met 480 485 490 495 gga agt ctg acc atc ctgaca gcc atc ctc acc ttg ttt ctc cca gag 1656 Gly Ser Leu Thr Ile Leu ThrAla Ile Leu Thr Leu Phe Leu Pro Glu 500 505 510 agc ttc ggt acc cca ctccca gac acc att gac cag atg cta aga gtc 1704 Ser Phe Gly Thr Pro Leu ProAsp Thr Ile Asp Gln Met Leu Arg Val 515 520 525 aaa gga atg aaa cac agaaaa act cca agt cac aca agg atg tta aaa 1752 Lys Gly Met Lys His Arg LysThr Pro Ser His Thr Arg Met Leu Lys 530 535 540 gat ggt caa gaa agg cccaca atc ctt aaa agc aca gcc ttc 1794 Asp Gly Gln Glu Arg Pro Thr Ile LeuLys Ser Thr Ala Phe 545 550 555 taacatcgct tccagtaagg gagaaactga agaggaa1831 <210> SEQ ID NO 5 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Artificial Synthesized Primer Sequence <400>SEQUENCE: 5 ctaatacgac tcactatagg gc 22 <210> SEQ ID NO 6 <211> LENGTH:21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: ArtificialSynthesized Primer Sequence <400> SEQUENCE: 6 tgtagcgtga agacgacaga a 21<210> SEQ ID NO 7 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Artificial Synthesized Primer Sequence <400>SEQUENCE: 7 tcgagcggcc gcccgggcag gt 22 <210> SEQ ID NO 8 <211> LENGTH:22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: ArtificialSynthesized Primer Sequence <400> SEQUENCE: 8 agggcgtggt gcggagggcg gt22 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Artificial Synthesized Primer Sequence <400>SEQUENCE: 9 cttttgagca agttcagcct 20 <210> SEQ ID NO 10 <211> LENGTH: 24<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: ArtificialSynthesized Primer Sequence <400> SEQUENCE: 10 agaggtggct tatgagtatttctt 24 <210> SEQ ID NO 11 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Artificial Synthesized PrimerSequence <400> SEQUENCE: 11 ccagggtttt cccagtcacg ac 22 <210> SEQ ID NO12 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Artificial Synthesized Primer Sequence <400> SEQUENCE: 12tcacacagga aacagctatg ac 22 <210> SEQ ID NO 13 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Artificial SynthesizedPrimer Sequence <400> SEQUENCE: 13 gtgctgttgg gctccttcat ttca 24 <210>SEQ ID NO 14 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Artificial Synthesized Primer Sequence <400>SEQUENCE: 14 agctgcatga agagaaggac actg 24 <210> SEQ ID NO 15 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Artificial Synthesized Primer Sequence <400> SEQUENCE: 15 agcatcctgtctccctactt cgtt 24 <210> SEQ ID NO 16 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Artificial SynthesizedPrimer Sequence <400> SEQUENCE: 16 gatggatccc ggacggtctt gggtcgcctg ctg33 <210> SEQ ID NO 17 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Artificial Synthesized Primer Sequence <400>SEQUENCE: 17 gatggatcca aatgctgcca catagttgga gat 33 <210> SEQ ID NO 18<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Artificial Synthesized Primer Sequence <400> SEQUENCE: 18gatggatcca tgggcatgca gacaggcttc agc 33 <210> SEQ ID NO 19 <211> LENGTH:33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: ArtificialSynthesized Primer Sequence <400> SEQUENCE: 19 gatggatcct tcctcttcagtttctccctt act 33 <210> SEQ ID NO 20 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Artificial SynthesizedPrimer Sequence <400> SEQUENCE: 20 cgcgccgaat cgctgaatcc tttc 24 <210>SEQ ID NO 21 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Artificial Synthesized Primer Sequence <400>SEQUENCE: 21 aggcttttga tttgttctgt tgag 24 <210> SEQ ID NO 22 <211>LENGTH: 553 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:22 Met Arg Asp Tyr Asp Glu Val Ile Ala Phe Leu Gly Glu Trp Gly Pro 1 510 15 Phe Gln Arg Leu Ile Phe Phe Leu Leu Ser Ala Ser Ile Ile Pro Asn 2025 30 Gly Phe Asn Gly Met Ser Val Val Phe Leu Ala Gly Thr Pro Glu His 3540 45 Arg Cys Leu Val Pro Asp Thr Val Asn Leu Ser Ser Ser Trp Arg Asn 5055 60 His Ser Ile Pro Leu Glu Thr Lys Asp Gly Arg Gln Val Pro Gln Ser 6570 75 80 Cys Arg Arg Tyr Arg Leu Ala Thr Ile Ala Asn Phe Ser Ala Met Gly85 90 95 Leu Glu Pro Gly Gln Asp Val Asp Leu Glu Gln Leu Glu Gln Glu Ser100 105 110 Cys Leu Asp Gly Trp Glu Tyr Asp Lys Asp Ile Phe Leu Ser ThrIle 115 120 125 Val Thr Glu Trp Asn Leu Val Cys Glu Asp Asp Trp Lys ThrPro Leu 130 135 140 Thr Thr Ser Leu Phe Phe Val Gly Val Leu Cys Gly SerPhe Val Ser 145 150 155 160 Gly Gln Leu Ser Asp Arg Phe Gly Arg Lys LysVal Leu Phe Ala Thr 165 170 175 Met Ala Val Gln Thr Gly Phe Ser Phe ValGln Ile Phe Ser Thr Asn 180 185 190 Trp Glu Met Phe Thr Val Leu Phe AlaIle Val Gly Met Gly Gln Ile 195 200 205 Ser Asn Tyr Val Val Ala Phe IleLeu Gly Thr Glu Ile Leu Ser Lys 210 215 220 Ser Val Arg Ile Ile Phe SerThr Leu Gly Val Cys Thr Phe Phe Ala 225 230 235 240 Ile Gly Tyr Met ValLeu Pro Leu Phe Ala Tyr Phe Ile Arg Asp Trp 245 250 255 Arg Met Leu LeuLeu Ala Leu Thr Leu Pro Gly Leu Phe Cys Val Pro 260 265 270 Leu Trp TrpPhe Ile Pro Glu Ser Pro Arg Trp Leu Ile Ser Gln Arg 275 280 285 Arg PheAla Glu Ala Glu Gln Ile Ile Gln Lys Ala Ala Lys Met Asn 290 295 300 SerIle Val Ala Pro Ala Gly Ile Phe Asp Pro Leu Glu Leu Gln Glu 305 310 315320 Leu Asn Ser Leu Lys Gln Gln Lys Val Ile Ile Leu Asp Leu Phe Arg 325330 335 Thr Arg Asn Ile Ala Thr Ile Thr Val Met Ala Val Met Leu Trp Met340 345 350 Leu Thr Ser Val Gly Tyr Phe Ala Leu Ser Leu Asn Val Pro AsnLeu 355 360 365 His Gly Asp Val Tyr Leu Asn Cys Phe Leu Ser Gly Leu IleGlu Val 370 375 380 Pro Ala Tyr Phe Thr Ala Trp Leu Leu Leu Arg Thr LeuPro Arg Arg 385 390 395 400 Tyr Ile Ile Ala Gly Val Leu Phe Trp Gly GlyGly Val Leu Leu Leu 405 410 415 Ile Gln Val Val Pro Glu Asp Tyr Asn PheVal Ser Ile Gly Leu Val 420 425 430 Met Leu Gly Lys Phe Gly Ile Thr SerAla Phe Ser Met Leu Tyr Val 435 440 445 Phe Thr Ala Glu Leu Tyr Pro ThrLeu Val Arg Asn Met Ala Val Gly 450 455 460 Ile Thr Ser Met Ala Ser ArgVal Gly Ser Ile Ile Ala Pro Tyr Phe 465 470 475 480 Val Tyr Leu Gly AlaTyr Asn Arg Leu Leu Pro Tyr Ile Leu Met Gly 485 490 495 Ser Leu Thr ValLeu Ile Gly Ile Ile Thr Leu Phe Phe Pro Glu Ser 500 505 510 Phe Gly ValThr Leu Pro Glu Asn Leu Glu Gln Met Gln Lys Val Arg 515 520 525 Gly PheArg Cys Gly Lys Lys Ser Thr Val Ser Val Asp Arg Glu Glu 530 535 540 SerPro Lys Val Leu Ile Thr Ala Phe 545 550 <210> SEQ ID NO 23 <211> LENGTH:2083 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (122)..(1780) <400> SEQUENCE: 23attcggcaca ggacggcgtg tttgacgagc cacctaggaa gatcccctca gcgcgccgaa 60tcgctgaatc ctttctctcc acccacctcc ctcacgcaag ctgaggagga gaggtggaaa 120 catg cgg gac tac gac gag gtg atc gcc ttc ctg ggc gag tgg ggg ccc 169 MetArg Asp Tyr Asp Glu Val Ile Ala Phe Leu Gly Glu Trp Gly Pro 1 5 10 15ttc cag cgc ctc atc ttc ttt ctg ctc agc gcc agc atc atc ccc aat 217 PheGln Arg Leu Ile Phe Phe Leu Leu Ser Ala Ser Ile Ile Pro Asn 20 25 30 ggcttc aat ggt atg tca gtc gtg ttc ctg gcg ggg acc ccc gag cac 265 Gly PheAsn Gly Met Ser Val Val Phe Leu Ala Gly Thr Pro Glu His 35 40 45 cgt tgcctg gtt cct gac act gtg aac ctg agc agc tcc tgg cgc aac 313 Arg Cys LeuVal Pro Asp Thr Val Asn Leu Ser Ser Ser Trp Arg Asn 50 55 60 cac agc atcccc ttg gag acg aag gac gga cga cag gtg cct cag agc 361 His Ser Ile ProLeu Glu Thr Lys Asp Gly Arg Gln Val Pro Gln Ser 65 70 75 80 tgc cgc cgctac cga ctg gcc acc atc gcc aac ttc tct gcg atg ggg 409 Cys Arg Arg TyrArg Leu Ala Thr Ile Ala Asn Phe Ser Ala Met Gly 85 90 95 ctg gag cca ggacag gac gtg gat ctg gag cag ctg gag cag gag agc 457 Leu Glu Pro Gly GlnAsp Val Asp Leu Glu Gln Leu Glu Gln Glu Ser 100 105 110 tgc ctg gat ggctgg gag tac gac aag gac atc ttc ctg tcc acc atc 505 Cys Leu Asp Gly TrpGlu Tyr Asp Lys Asp Ile Phe Leu Ser Thr Ile 115 120 125 gtg aca gag tggaat ctg gtg tgt gag gat gac tgg aag aca ccc ctc 553 Val Thr Glu Trp AsnLeu Val Cys Glu Asp Asp Trp Lys Thr Pro Leu 130 135 140 acc acc tcc ctgttc ttc gta ggc gtt ctc tgc ggc tcc ttc gtg tct 601 Thr Thr Ser Leu PhePhe Val Gly Val Leu Cys Gly Ser Phe Val Ser 145 150 155 160 ggg cag ctgtca gac agg ttt ggc agg aag aaa gtc ctc ttt gca acc 649 Gly Gln Leu SerAsp Arg Phe Gly Arg Lys Lys Val Leu Phe Ala Thr 165 170 175 atg gct gtgcag act gga ttc agc ttc gtg cag att ttc tca acc aac 697 Met Ala Val GlnThr Gly Phe Ser Phe Val Gln Ile Phe Ser Thr Asn 180 185 190 tgg gag atgttc act gtg ttg ttt gcc att gtg ggc atg ggc cag atc 745 Trp Glu Met PheThr Val Leu Phe Ala Ile Val Gly Met Gly Gln Ile 195 200 205 tcc aac tacgtg gtg gcc ttc ata cta gga act gaa atc ctg agc aag 793 Ser Asn Tyr ValVal Ala Phe Ile Leu Gly Thr Glu Ile Leu Ser Lys 210 215 220 tcg gtt cgcatc atc ttc tcc aca tta gga gtc tgt aca ttt ttt gca 841 Ser Val Arg IleIle Phe Ser Thr Leu Gly Val Cys Thr Phe Phe Ala 225 230 235 240 atc ggctac atg gtc ctg ccg ctg ttt gca tac ttc atc aga gac tgg 889 Ile Gly TyrMet Val Leu Pro Leu Phe Ala Tyr Phe Ile Arg Asp Trp 245 250 255 agg atgctg ctg ctg gcc ctg aca ctg cct ggc ctg ttc tgt gtt ccc 937 Arg Met LeuLeu Leu Ala Leu Thr Leu Pro Gly Leu Phe Cys Val Pro 260 265 270 ctg tggtgg ttt att cca gaa tct ccc cgg tgg ctg ata tcc cag agg 985 Leu Trp TrpPhe Ile Pro Glu Ser Pro Arg Trp Leu Ile Ser Gln Arg 275 280 285 aga tttgca gag gcc gaa cag atc atc cag aaa gcc gca aag atg aac 1033 Arg Phe AlaGlu Ala Glu Gln Ile Ile Gln Lys Ala Ala Lys Met Asn 290 295 300 agc atcgtg gcg cca gca ggg ata ttc gat cct cta gag cta cag gag 1081 Ser Ile ValAla Pro Ala Gly Ile Phe Asp Pro Leu Glu Leu Gln Glu 305 310 315 320 ctaaac tcc ttg aag cag cag aaa gtc ata atc ctg gac ctg ttc agg 1129 Leu AsnSer Leu Lys Gln Gln Lys Val Ile Ile Leu Asp Leu Phe Arg 325 330 335 actcgg aac att gcc acc ata acc gtg atg gct gtg atg ctg tgg atg 1177 Thr ArgAsn Ile Ala Thr Ile Thr Val Met Ala Val Met Leu Trp Met 340 345 350 ctaacc tca gtg ggt tac ttt gct ctg tct ctc aat gtt cct aat tta 1225 Leu ThrSer Val Gly Tyr Phe Ala Leu Ser Leu Asn Val Pro Asn Leu 355 360 365 catgga gat gtc tac ctg aac tgc ttc ctc tct ggc ctg att gaa gtt 1273 His GlyAsp Val Tyr Leu Asn Cys Phe Leu Ser Gly Leu Ile Glu Val 370 375 380 ccagct tac ttc aca gcc tgg ctg cta ctg cga acc ctg cca cgg aga 1321 Pro AlaTyr Phe Thr Ala Trp Leu Leu Leu Arg Thr Leu Pro Arg Arg 385 390 395 400tat att ata gct ggg gtg cta ttc tgg gga gga ggt gtg ctt ctc ttg 1369 TyrIle Ile Ala Gly Val Leu Phe Trp Gly Gly Gly Val Leu Leu Leu 405 410 415atc caa gtg gta cct gaa gat tat aac ttt gtg tcc att gga ctg gtg 1417 IleGln Val Val Pro Glu Asp Tyr Asn Phe Val Ser Ile Gly Leu Val 420 425 430atg ctg ggg aaa ttt ggg atc acc tct gcc ttc tcc atg ttg tat gtc 1465 MetLeu Gly Lys Phe Gly Ile Thr Ser Ala Phe Ser Met Leu Tyr Val 435 440 445ttc act gcg gag ctc tac cca acc ctg gtc agg aac atg gct gtg ggc 1513 PheThr Ala Glu Leu Tyr Pro Thr Leu Val Arg Asn Met Ala Val Gly 450 455 460atc acc tcc atg gcc tct cgg gtg ggc agc atc att gcc ccc tat ttc 1561 IleThr Ser Met Ala Ser Arg Val Gly Ser Ile Ile Ala Pro Tyr Phe 465 470 475480 gtt tac ctg ggc gcc tat aac aga ctc cta ccc tac atc ctc atg ggc 1609Val Tyr Leu Gly Ala Tyr Asn Arg Leu Leu Pro Tyr Ile Leu Met Gly 485 490495 agt ctg act gtc ctc att gga atc atc acg ctt ttt ttc cct gaa agt 1657Ser Leu Thr Val Leu Ile Gly Ile Ile Thr Leu Phe Phe Pro Glu Ser 500 505510 ttt gga gtg act cta cca gag aac ttg gag cag atg cag aaa gtg aga 1705Phe Gly Val Thr Leu Pro Glu Asn Leu Glu Gln Met Gln Lys Val Arg 515 520525 ggg ttc aga tgt ggg aaa aaa tca aca gtc tca gtg gac aga gaa gaa 1753Gly Phe Arg Cys Gly Lys Lys Ser Thr Val Ser Val Asp Arg Glu Glu 530 535540 agc ccc aag gtt cta ata act gca ttc taacgaggtt tccaaggcac 1800 SerPro Lys Val Leu Ile Thr Ala Phe 545 550 ttggcaaact gaaaagcaga tgtatacaatgagcagggtg tgatagagca agcctgcaat 1860 cccagcgctc ttggggtgga gacagaagatcaggagttca aggtcatcct tggctacagc 1920 aggagtgtaa gaccagcctg tcttaccacaagcaaccctg tctcaacaga acaaatcaaa 1980 agccttttct gctgaaaggg attaacagaaacaatgagca ccaaactgga cttgtggaga 2040 aatgcacact atctcatgaa ttctgggccactcttccaga tgg 2083 <210> SEQ ID NO 24 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Artificial SynthesizedPrimer Sequence <400> SEQUENCE: 24 cccatgccaa caaggacaaa aagc 24 <210>SEQ ID NO 25 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Artificial Synthesized Primer Sequence <400>SEQUENCE: 25 acagaacaga aaagccctca gtca 24 <210> SEQ ID NO 26 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Artificial Synthesized Primer Sequence <400> SEQUENCE: 26 tgtttttcgtgggtgtgctg atgg 24 <210> SEQ ID NO 27 <211> LENGTH: 557 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 27 Met Arg Asp Tyr Asp GluVal Thr Ala Phe Leu Gly Glu Trp Gly Pro 1 5 10 15 Phe Gln Arg Leu IlePhe Phe Leu Leu Ser Ala Ser Ile Ile Pro Asn 20 25 30 Gly Phe Asn Gly MetSer Ile Val Phe Leu Ala Gly Thr Pro Glu His 35 40 45 Arg Cys Leu Val ProHis Thr Val Asn Leu Ser Ser Ala Trp Arg Asn 50 55 60 His Ser Ile Pro LeuGlu Thr Lys Asp Gly Arg Gln Val Pro Gln Lys 65 70 75 80 Cys Arg Arg TyrArg Leu Ala Thr Ile Ala Asn Phe Ser Glu Leu Gly 85 90 95 Leu Glu Pro GlyArg Asp Val Asp Leu Glu Gln Leu Glu Gln Glu Ser 100 105 110 Cys Leu AspGly Trp Glu Tyr Asp Lys Asp Val Phe Leu Ser Thr Ile 115 120 125 Val ThrGlu Trp Asp Leu Val Cys Lys Asp Asp Trp Lys Ala Pro Leu 130 135 140 ThrThr Ser Leu Phe Phe Val Gly Val Leu Met Gly Ser Phe Ile Ser 145 150 155160 Gly Gln Leu Ser Asp Arg Phe Gly Arg Lys Asn Val Leu Phe Leu Thr 165170 175 Met Gly Met Gln Thr Gly Phe Ser Phe Leu Gln Val Phe Ser Val Asn180 185 190 Phe Glu Met Phe Thr Val Leu Phe Val Leu Val Gly Met Gly GlnIle 195 200 205 Ser Asn Tyr Val Ala Ala Phe Val Leu Gly Thr Glu Ile LeuSer Lys 210 215 220 Ser Ile Arg Ile Ile Phe Ala Thr Leu Gly Val Cys IlePhe Tyr Ala 225 230 235 240 Phe Gly Phe Met Val Leu Pro Leu Phe Ala TyrPhe Ile Arg Asp Trp 245 250 255 Arg Met Leu Leu Leu Ala Leu Thr Val ProGly Val Leu Cys Gly Ala 260 265 270 Leu Trp Trp Phe Ile Pro Glu Ser ProArg Trp Leu Ile Ser Gln Gly 275 280 285 Arg Ile Lys Glu Ala Glu Val IleIle Arg Lys Ala Ala Lys Ile Asn 290 295 300 Gly Ile Val Ala Pro Ser ThrIle Phe Asp Pro Ser Glu Leu Gln Asp 305 310 315 320 Leu Asn Ser Thr LysPro Gln Leu His His Ile Tyr Asp Leu Ile Arg 325 330 335 Thr Arg Asn IleArg Val Ile Thr Ile Met Ser Ile Ile Leu Trp Leu 340 345 350 Thr Ile SerVal Gly Tyr Phe Gly Leu Ser Leu Asp Thr Pro Asn Leu 355 360 365 His GlyAsp Ile Tyr Val Asn Cys Phe Leu Leu Ala Ala Val Glu Val 370 375 380 ProAla Tyr Val Leu Ala Trp Leu Leu Leu Gln Tyr Leu Pro Arg Arg 385 390 395400 Tyr Ser Ile Ser Ala Ala Leu Phe Leu Gly Gly Ser Val Leu Leu Phe 405410 415 Met Gln Leu Val Pro Ser Glu Leu Phe Tyr Leu Ser Thr Ala Leu Val420 425 430 Met Val Gly Lys Phe Gly Ile Thr Ser Ala Tyr Ser Met Val TyrVal 435 440 445 Tyr Thr Ala Glu Leu Tyr Pro Thr Val Val Arg Asn Met GlyVal Gly 450 455 460 Val Ser Ser Thr Ala Ser Arg Leu Gly Ser Ile Leu SerPro Tyr Phe 465 470 475 480 Val Tyr Leu Gly Ala Tyr Asp Arg Phe Leu ProTyr Ile Leu Met Gly 485 490 495 Ser Leu Thr Ile Leu Thr Ala Ile Leu ThrLeu Phe Phe Pro Glu Ser 500 505 510 Phe Gly Val Pro Leu Pro Asp Thr IleAsp Gln Met Leu Arg Val Lys 515 520 525 Gly Ile Lys Gln Trp Gln Ile GlnSer Gln Thr Arg Met Gln Lys Asp 530 535 540 Gly Glu Glu Ser Pro Thr ValLeu Lys Ser Thr Ala Phe 545 550 555 <210> SEQ ID NO 28 <211> LENGTH:1888 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (60)..(1730) <400> SEQUENCE: 28 ctcccgcgccacggtgtccc cttattccca tacgggcgct gtgggaggct gaggacggc 59 atg cgg gac tacgac gag gtg acc gcc ttc cta ggc gag tgg ggg ccc 107 Met Arg Asp Tyr AspGlu Val Thr Ala Phe Leu Gly Glu Trp Gly Pro 1 5 10 15 ttc cag cgc ctcatc ttc ttc ctg ctc agc gcc agc atc atc ccc aat 155 Phe Gln Arg Leu IlePhe Phe Leu Leu Ser Ala Ser Ile Ile Pro Asn 20 25 30 ggc ttc aat ggt atgtcc atc gtg ttc ctg gcg ggg acc ccg gag cac 203 Gly Phe Asn Gly Met SerIle Val Phe Leu Ala Gly Thr Pro Glu His 35 40 45 cgt tgc ctt gtg cct cacacc gtg aac ctg agc agc gcg tgg cgc aac 251 Arg Cys Leu Val Pro His ThrVal Asn Leu Ser Ser Ala Trp Arg Asn 50 55 60 cac agt atc ccg ttg gag acgaag gac gga cga cag gtg cct cag aaa 299 His Ser Ile Pro Leu Glu Thr LysAsp Gly Arg Gln Val Pro Gln Lys 65 70 75 80 tgc cgc cgc tac cga ctg gccacc atc gcc aac ttc tct gag cta ggg 347 Cys Arg Arg Tyr Arg Leu Ala ThrIle Ala Asn Phe Ser Glu Leu Gly 85 90 95 ctg gag ccg ggg cgg gac gtg gacctg gag cag ctg gag cag gag agc 395 Leu Glu Pro Gly Arg Asp Val Asp LeuGlu Gln Leu Glu Gln Glu Ser 100 105 110 tgc ctg gat ggc tgg gag tac gacaag gac gtc ttc ctg tcc acc atc 443 Cys Leu Asp Gly Trp Glu Tyr Asp LysAsp Val Phe Leu Ser Thr Ile 115 120 125 gtg aca gag tgg gac ctg gtg tgtaag gat gac tgg aaa gcc cca ctc 491 Val Thr Glu Trp Asp Leu Val Cys LysAsp Asp Trp Lys Ala Pro Leu 130 135 140 acc acc tcc ttg ttt ttc gtg ggtgtg ctg atg ggc tcc ttc att tca 539 Thr Thr Ser Leu Phe Phe Val Gly ValLeu Met Gly Ser Phe Ile Ser 145 150 155 160 gga cag ctc tca gac agg tttggt cgc aag aat gtg ctg ttt ttg acc 587 Gly Gln Leu Ser Asp Arg Phe GlyArg Lys Asn Val Leu Phe Leu Thr 165 170 175 atg ggc atg cag act ggc ttcagc ttc ctg cag gtc ttc tct gtg aac 635 Met Gly Met Gln Thr Gly Phe SerPhe Leu Gln Val Phe Ser Val Asn 180 185 190 ttc gag atg ttt aca gtg cttttt gtc ctt gtt ggc atg ggt cag atc 683 Phe Glu Met Phe Thr Val Leu PheVal Leu Val Gly Met Gly Gln Ile 195 200 205 tcc aac tac gtg gca gca tttgtc ctg gga aca gaa att ctt tcc aag 731 Ser Asn Tyr Val Ala Ala Phe ValLeu Gly Thr Glu Ile Leu Ser Lys 210 215 220 tca att cga att ata ttc gccacc tta gga gtt tgc ata ttt tat gcg 779 Ser Ile Arg Ile Ile Phe Ala ThrLeu Gly Val Cys Ile Phe Tyr Ala 225 230 235 240 ttt ggc ttc atg gtg ctgcca ctg ttt gca tac ttc atc aga gac tgg 827 Phe Gly Phe Met Val Leu ProLeu Phe Ala Tyr Phe Ile Arg Asp Trp 245 250 255 agg atg ctg ctg ctg gcgctc act gtg cca ggg gtg cta tgt ggg gct 875 Arg Met Leu Leu Leu Ala LeuThr Val Pro Gly Val Leu Cys Gly Ala 260 265 270 ctc tgg tgg ttc atc cctgag tcc cca cga tgg ctc atc tct caa ggc 923 Leu Trp Trp Phe Ile Pro GluSer Pro Arg Trp Leu Ile Ser Gln Gly 275 280 285 cga att aaa gag gca gaggtg atc atc cgc aaa gct gcc aaa atc aat 971 Arg Ile Lys Glu Ala Glu ValIle Ile Arg Lys Ala Ala Lys Ile Asn 290 295 300 ggg att gtt gca cct tccact atc ttc gat cca agt gag tta caa gac 1019 Gly Ile Val Ala Pro Ser ThrIle Phe Asp Pro Ser Glu Leu Gln Asp 305 310 315 320 tta aat tct acg aagcct cag ttg cac cac att tat gat ctg atc cga 1067 Leu Asn Ser Thr Lys ProGln Leu His His Ile Tyr Asp Leu Ile Arg 325 330 335 aca cgg aat atc agggtc atc acc atc atg tct ata atc ctg tgg ctg 1115 Thr Arg Asn Ile Arg ValIle Thr Ile Met Ser Ile Ile Leu Trp Leu 340 345 350 acc ata tca gtg ggctat ttt gga cta tct ctt gac act cct aac ttg 1163 Thr Ile Ser Val Gly TyrPhe Gly Leu Ser Leu Asp Thr Pro Asn Leu 355 360 365 cat ggg gac atc tatgtg aac tgc ttc cta ctg gcg gct gtt gaa gtc 1211 His Gly Asp Ile Tyr ValAsn Cys Phe Leu Leu Ala Ala Val Glu Val 370 375 380 cca gcc tat gtg ctggcc tgg ctg ttg ttg cag tac ttg ccc cgg cga 1259 Pro Ala Tyr Val Leu AlaTrp Leu Leu Leu Gln Tyr Leu Pro Arg Arg 385 390 395 400 tat tct atc tcggct gcc ctt ttc ctg ggt ggc agt gtc ctt ctc ttc 1307 Tyr Ser Ile Ser AlaAla Leu Phe Leu Gly Gly Ser Val Leu Leu Phe 405 410 415 atg cag ctg gtgcct tca gaa ttg ttt tac ttg tcc act gcc ctg gtg 1355 Met Gln Leu Val ProSer Glu Leu Phe Tyr Leu Ser Thr Ala Leu Val 420 425 430 atg gtg ggg aagttt gga atc acc tct gcc tac tcc atg gtc tat gtg 1403 Met Val Gly Lys PheGly Ile Thr Ser Ala Tyr Ser Met Val Tyr Val 435 440 445 tac aca gct gagctg tac ccc act gtg gtc aga aac atg ggt gtg ggg 1451 Tyr Thr Ala Glu LeuTyr Pro Thr Val Val Arg Asn Met Gly Val Gly 450 455 460 gtc agc tcc acagca tcc cgc ctt ggc agc atc ctg tct ccc tac ttt 1499 Val Ser Ser Thr AlaSer Arg Leu Gly Ser Ile Leu Ser Pro Tyr Phe 465 470 475 480 gtt tac ctaggt gcc tat gat cgc ttc ctg cct tat att ctc atg gga 1547 Val Tyr Leu GlyAla Tyr Asp Arg Phe Leu Pro Tyr Ile Leu Met Gly 485 490 495 agt ctg accatc ctg aca gct atc ctc acc ttg ttc ttc cct gag agc 1595 Ser Leu Thr IleLeu Thr Ala Ile Leu Thr Leu Phe Phe Pro Glu Ser 500 505 510 ttt ggt gtccct ctc cca gat acc att gac cag atg cta agg gtc aaa 1643 Phe Gly Val ProLeu Pro Asp Thr Ile Asp Gln Met Leu Arg Val Lys 515 520 525 gga ata aaacag tgg caa atc caa agc cag aca aga atg caa aaa gat 1691 Gly Ile Lys GlnTrp Gln Ile Gln Ser Gln Thr Arg Met Gln Lys Asp 530 535 540 ggt gaa gaaagc cca aca gtc cta aag agc aca gcc ttc taacaccctg 1740 Gly Glu Glu SerPro Thr Val Leu Lys Ser Thr Ala Phe 545 550 555 tccagaaggc aaaaaactgattggaaacct tcatgttgtc agaaatgctc tccatgactg 1800 agggcttttc tgttctgttaaccttgtgtc taacatgctc atggattggg gcatctgtcc 1860 tggagagtca ccttcctctagggacacc 1888 <210> SEQ ID NO 29 <211> LENGTH: 44 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Artificial Synthesized AdapterSequence <400> SEQUENCE: 29 ctaatacgac tcactatagg gctcgagcgg ccgcccgggcaggt 44 <210> SEQ ID NO 30 <211> LENGTH: 43 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Artificial Synthesized AdapterSequencence <400> SEQUENCE: 30 tgtagcgtga agacgacaga aagggcgtggtgcggagggc ggt 43 <210> SEQ ID NO 31 <211> LENGTH: 10 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Artificial SynthesizedAdapter Sequence <400> SEQUENCE: 31 acctgcccgg 10 <210> SEQ ID NO 32<211> LENGTH: 11 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Artificial Synthesized Adapter Sequence <400> SEQUENCE: 32accgccctcc g 11 <210> SEQ ID NO 33 <211> LENGTH: 14 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Exemplary motif <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: 1 <223> OTHER INFORMATION: Xaa = Leu, Ile, Val, Met,Ser, Thr, Ala, or Gly <220> FEATURE: <221> NAME/KEY: VARIANT <222>LOCATION: 2 <223> OTHER INFORMATION: Xaa = Leu, Ile, Val, Met, Phe, Ser,Ala, or Gly <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 3, 6,11-13 <223> OTHER INFORMATION: Xaa = any amino acid <220> FEATURE: <221>NAME/KEY: VARIANT <222> LOCATION: 4 <223> OTHER INFORMATION: Xaa = Leu,Ile, Val, Met, Ser, or Ala <220> FEATURE: <221> NAME/KEY: VARIANT <222>LOCATION: 5 <223> OTHER INFORMATION: Xaa = Asp, or Glu <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: 7 <223> OTHER INFORMATION: Xaa =Leu, Ile, Val, Met, Phe, Tyr, Trp, or Ala <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: 10 <223> OTHER INFORMATION: Xaa = Arg, or Lys<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 14 <223> OTHERINFORMATION: Xaa = Gly, Ser, Thr, or Ala <400> SEQUENCE: 33 Xaa Xaa XaaXaa Xaa Xaa Xaa Gly Arg Xaa Xaa Xaa Xaa Xaa 1 5 10

What is claimed is:
 1. A substantially pure polypeptide comprising anamino acid sequence at least 70% identical to any one of SEQ ID NOs:1,3, 22, or 27, wherein the polypeptide is a transporter of an organiccation.
 2. The polypeptide of claim 1, wherein the amino acid sequenceis at least 80% identical to any one of SEQ ID NOs:1, 3, 22, or
 27. 3.The polypeptide of claim 1, wherein the amino acid sequence is at least90% identical to any one of SEQ ID NOs:1, 3, 22, or
 27. 4. Thepolypeptide of claim 1, wherein the amino acid sequence is at least 95%identical to any one of SEQ ID NOs:1, 3, 22, or
 27. 5. A substantiallypure polypeptide comprising the sequence of any one of SEQ ID NOs:1, 3,22, or
 27. 6. A substantially pure polypeptide comprising the amino acidsequence of any one of SEQ ID NOs:1, 3, 22, or 27, with up to 30conservative amino acid substitutions, wherein the polypeptide is atransporter of an organic cation.
 7. A substantially pure polypeptideencoded by a nucleic acid that hybridizes under stringent conditions toa probe the sequence of which consists of any one of SEQ ID NOs:2, 4,23, or 28, wherein the polypeptide is a transporter of an organiccation.
 8. An isolated nucleic acid encoding the polypeptide of claim 1.9. An isolated nucleic acid encoding the polypeptide of claim
 5. 10. Anisolated nucleic acid encoding the polypeptide of claim
 6. 11. Anisolated nucleic acid comprising a strand that hybridizes understringent conditions to a single stranded probe, the sequence of whichconsists of any one of SEQ ID NOs:2, 4, 23, or 28, or the complement ofany one of SEQ ID NOs:2, 4, 23, or
 28. 12. The isolated nucleic acid ofclaim 11, wherein the nucleic acid encodes a polypeptide that is atransporter of an organic cation.
 13. The nucleic acid of claim 12,wherein the amino acid sequence of the polypeptide comprises any one ofSEQ ID NOs: 1, 3, 22, or
 27. 14. The nucleic acid of claim 11, whereinthe strand is at least 15 nucleotides in length.
 15. The nucleic acid ofclaim 14, wherein the nucleic acid is an antisense nucleic acid thatinhibits expression of a polypeptide comprising any one of SEQ ID NOs:1, 3, 22, or
 27. 16. A vector comprising the nucleic acid of claim 8.17. A vector comprising the nucleic acid of claim
 9. 18. A vectorcomprising the nucleic acid of claim
 10. 19. A vector comprising thenucleic acid of claim
 11. 20. A vector comprising the nucleic acid ofclaim
 12. 21. A cultured host cell comprising the nucleic acid of claim8.
 22. A cultured host cell comprising the nucleic acid of claim
 9. 23.A cultured host cell comprising the nucleic acid of claim
 10. 24. Acultured host cell comprising the nucleic acid of claim
 11. 25. Acultured host cell comprising the nucleic acid of claim
 12. 26. Anantibody that specifically binds to the polypeptide of claim
 1. 27. Amethod of producing a polypeptide, the method comprising isolating thepolypeptide from the cultured host cell of claim
 21. 28. The polypeptideof claim 1, wherein the polypeptide comprises the sequenceXaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Gly-Arg-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12,wherein Xaa1 is Leu, Ile, Val, Met, Ser, Thr, Ala, or Gly; Xaa2 is Leu,Ile, Val, Met, Phe, Ser, Ala, or Gly; Xaa3 is any amino acid; Xaa4 isLeu, Ile, Val, Met, Ser, Ala Xaa5 is Asp or Glu; Xaa6 is any amino acid;Xaa7 is Leu, Ile, Val, Met, Phe, Tyr, Trp, or Ala; Xaa8 is Arg or Lys;Xaa9 is any amino acid; Xaa10 is any amino acid; Xaa11 is any aminoacid; and Xaa12 is Gly, Ser, Thr, or Ala.